Compositions and methods for treating inflammatory diseases

ABSTRACT

The invention provides compositions and methods for treating inflammatory diseases, such as cardiac or hepatic inflammatory diseases, involving the use of parasite-derived neurotrophic factor (PDNF), or fragment of PDNF.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/768,157, filed on Aug. 14, 2015, which is the U.S. NationalStage Application, pursuant to 35 U.S.C. §371, of International PCTApplication No. PCT/US2014/29323, filed Mar. 14, 2014, designating theUnited States and published in English, which claims priority to andbenefit of U.S. Provisional Application No. 61/918,260, filed Dec. 19,2013, and U.S. Provisional Application No. 61/784,814, filed Mar. 14,2013, all of which are incorporated herein by reference in theirentireties.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with government support under Grant Nos.NS040574, NS42960, and AI09738 awarded by the National Institutes ofHealth (NIH). The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Inflammation is a mechanism that protects mammals from invadingpathogens and other insults, whether from the environment or from insidethe body. However, while transient inflammation is necessary to protecta mammal from infection, uncontrolled inflammation causes tissue damageand is the underlying cause of many illnesses.

Several types of cardiomyopathies are characterized by excessivetissue-destroying inflammation and fibrosis that leads to cardiacfailure and sudden death. Heart failure is a major economic burdenworldwide, estimated to cost per year about $34.4 billion. Orthotopicheart transplant is the only available cure but it is not practicalbecause of the relative absence of donor hearts. Hence, identifyingregenerative strategies for heart failure is a most urgent clinicalneed.

Because current methods for treating or preventing inflammation areinadequate, compositions and methods for treating or preventinginflammation are urgently required. Such compositions and methods areuseful for the prevention and/or treatment of inflammatory diseases anddisorders (e.g., in heart, liver, and gastrointestinal tract).

SUMMARY OF THE INVENTION

As described below, the present invention features compositionscomprising PDNF, or a fragment thereof, that provide for the preventionand/or treatment of inflammatory conditions in non-neuronal tissues(e.g., heart, liver, and gastrointestinal tract). The invention alsoprovides compositions featuring PDNF, or a fragment thereof, and methodsfor using such compositions for the proliferation and/or mobilization ofa stem cell (e.g., cardiac stem cell) or progenitor cell (e.g., hepaticprogenitor cell).

In one aspect, the invention provides a method of decreasinginflammation in a non-neuronal tissue of a subject, involvingadministering to the subject soluble parasite-derived neurotrophicfactor (sPDNF), or fragment thereof, in an amount effective to decreaseinflammation in the non-neuronal tissue.

In another aspect, the invention provides a method of decreasinginflammation in a cardiac, liver, pancreas, or gastrointestinal tissueof a subject, involving administering to the subject solubleparasite-derived neurotrophic factor (sPDNF), or a fragment thereof, inan amount effective to decrease inflammation in the cardiac, liver,pancreatic, or gastrointestinal tissue.

In still another aspect, the invention provides a method of increasingexpression of an anti-inflammatory factor in a non-neuronal cell ortissue, involving contacting the non-neuronal cell or tissue withsoluble parasite-derived neurotrophic factor (sPDNF), or a fragmentthereof, in an amount effective to increase expression of ananti-inflammatory factor in the non-neuronal cell or tissue.

In one aspect, the invention provides a method of increasing stem cellnumber, involving contacting a non-neuronal cell or tissue with solubleparasite-derived neurotrophic factor (sPDNF), or fragment thereof.

In another aspect, the invention provides a method of increasing stemcell mobilization, involving contacting a non-neuronal cell or tissuewith soluble parasite-derived neurotrophic factor (sPDNF), or fragmentthereof.

In yet another aspect, the invention provides a method of increasingstem cell proliferation, involving contacting a non-neuronal cell ortissue with soluble parasite-derived neurotrophic factor (sPDNF), orfragment thereof.

In one aspect, the invention provides a method of treating or preventinga cardiac inflammatory disease in a subject in need thereof, involvingadministering to the subject a therapeutically effective amount ofsoluble parasite-derived neurotrophic factor (sPDNF), or fragmentthereof.

In another aspect, the invention provides a method of treating orpreventing a hepatic inflammatory disease in a subject in need thereof,involving administering to the subject a therapeutically effectiveamount of soluble parasite-derived neurotrophic factor (sPDNF), orfragment thereof.

In still another aspect, the invention provides a method of treating orpreventing a pancreatic inflammatory disease in a subject in needthereof, involving administering to the subject a therapeuticallyeffective amount of soluble parasite-derived neurotrophic factor(sPDNF), or fragment thereof.

In yet another aspect, the invention provides a method of treating orpreventing an inflammatory disease of the gastrointestinal (GI) tract ina subject in need thereof, involving administering to the subject atherapeutically effective amount of soluble parasite-derivedneurotrophic factor (sPDNF), or fragment thereof.

In various embodiments of any aspect delineated herein, sPDNF, orfragment of sPDNF, has at least 85%, 90%, 95%, or 99% identity to theamino acid sequence set forth in residues 1-588 of SEQ ID NO: 2. Invarious embodiments of any aspect delineated herein, sPDNF, or fragmentof sPDNF, comprises an amino acid sequence that is selected from thegroup consisting of: (a) the amino acid sequence set forth in residues33-666 of SEQ ID NO: 2; (b) the amino acid sequence set forth inresidues 1 to 596 of SEQ ID NO: 4; and (c) an amino acid sequence thatis at least 85% identical to any one of (a) or (b).

In various embodiments of any aspect delineated herein, where thenon-neuronal cell or tissue is in vivo or ex vivo. In variousembodiments of any aspect delineated herein, the non-neuronal cell or isa cardiac, hepatic, pancreatic, and/or gastrointestinal cell or tissue.In various embodiments, the anti-inflammatory factor is one or more ofIL1-Ra, TSG-6 and COX-2. In various embodiments of any aspect delineatedherein, the stem cell is in vivo or ex vivo. In various embodiments, thestem cell expresses Sca-1 and/or c-Kit. In particular embodiments, thestem cell is a cardiac, hepatic, pancreatic, and/or gastrointestinalstem cell.

In various embodiments of any aspect delineated herein, the non-neuronalcell or tissue is in a subject. In various embodiments of any aspectdelineated herein, the subject (e.g., human subject) has or is at riskof having an inflammatory disease. In various embodiments, theinflammatory disease is a cardiac inflammatory disease, hepaticinflammatory disease, pancreatic inflammatory disease, and/orinflammatory disease of the gastrointestinal (GI) tract. In particularembodiments, the cardiac inflammatory disease is myocarditis,cardiomyopathy, endocarditis, and/or pericarditis. In particularembodiments, the hepatic inflammatory disease is hepatitis and/orcirrhosis. In particular embodiments, the pancreatic inflammatorydisease is type 1 or type 2 diabetes. In particular embodiments, theinflammatory disease of the GI tract is inflammatory bowel disease(IBD), irritable bowel syndrome, ileitis, chronic inflammatoryintestinal disease, celiac disease, Crohn's disease, and/or ulcerativecolitis. In various embodiments of any aspect delineated herein, thesubject does not have Chagas disease.

In various embodiments of any aspect delineated herein, sPDNF, orfragment thereof, is administered parenterally, intraperitoneally,subcutaneously, or intravenously. In various embodiments of any aspectdelineated herein, the method further involves administering ananti-inflammatory agent.

Compositions and articles defined by the invention were isolated orotherwise manufactured in connection with the examples provided below.Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “anti-inflammatory” is meant an agent that reduces the severity orsymptoms of an inflammatory reaction in a tissue. An inflammatoryreaction within tissue is generally characterized by leukocyteinfiltration, edema, redness, pain, and/or neovascularization.Inflammation can also be measured by analyzing levels of cytokines orany other inflammatory marker.

The term “anti-inflammatory amount” as used herein means the amountwhich reduces, alleviates, or inhibits inflammation in the tissue orbody.

By “decreases” is meant a negative alteration of at least 10%, 25%, 50%,75%, 100%, or more.

“Detect” refers to identifying the presence, absence or amount of theanalyte to be detected.

By “effective amount” is meant the amount of an agent required toameliorate the symptoms of a disease relative to an untreated patient.The effective amount of active agent(s) used to practice the presentinvention for therapeutic treatment of a disease varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. Ultimately, the attending physician or veterinarian willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

By “identity” is meant the amino acid or nucleic acid sequence identitybetween a sequence of interest and a reference sequence. Sequenceidentity is typically measured using sequence analysis software (forexample, Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOXprograms). Such software matches identical or similar sequences byassigning degrees of homology to various substitutions, deletions,and/or other modifications. Conservative substitutions typically includesubstitutions within the following groups: glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid, asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. In an exemplary approach to determining the degree ofidentity, a BLAST program may be used, with a probability score betweene⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

By “increases” is meant a positive alteration of at least 10%, 25%, 50%,75%, 100%, or more.

As used herein, the phrase “inflammatory disease” or “inflammatorydisorder” disorder” includes any disease, disorder, or condition that iscaused by or related to an inflammatory process within one or moretissue or serum of the body of a mammal. In certain embodiments, theinflammatory disease is mediated by the interleukin-1 (IL-1) receptor.In particular embodiments, the inflammatory disease or disorder is in anon-neuronal tissue, including but not limited to atherosclerosis,diabetes (e.g., diabetes Type 1 and 2), arthritis, cardiovasculardisease, and organ damage, such as liver damage for example.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation. A “purified” or“biologically pure” protein is sufficiently free of other materials suchthat any impurities do not materially affect the biological propertiesof the protein or cause other adverse consequences. That is, a nucleicacid or peptide of this invention is purified if it is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Purity and homogeneity aretypically determined using analytical chemistry techniques, for example,polyacrylamide gel electrophoresis or high performance liquidchromatography. The term “purified” can denote that a nucleic acid orprotein gives rise to essentially one band in an electrophoretic gel.For a protein that can be subjected to modifications, for example,phosphorylation or glycosylation, different modifications may give riseto different isolated proteins, which can be separately purified.

By “hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences (e.g., genes listed in Tables 1and 2), or portions thereof, under various conditions of stringency.(See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol.152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and most preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and most preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred: embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/mldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity that is associated with a disease ordisorder.

By “neuronal” is meant any cell or tissue that includes neurons andneural cells related to the nervous system, including glial cells andSchwann cells

By “non-neuronal” is meant any cell or tissue excluding neuronal cellsor tissues (e.g., neurons and cells related to the nervous system).related to the nervous system. In particular embodiments a non-neuronalcell or tissue includes, for example, cardiac, hepatic, pancreatic, andgastrointestinal cells and tissues.

By “parasite-derived neurotrophic factor (PDNF) polypeptide” is meant apolypeptide or fragment thereof having at least about 85%, 90%, 95%,96%, 97%, 98%, or 99% amino acid identity to NCBI Accession No. NP001387 and that specifically binds a Trk receptor (e.g., TrkA, TrkC).Exemplary PDNF polypeptide sequences are provided in FIGS. 1B and 1D.

By “soluble parasite-derived neurotrophic factor (sPDNF) polypeptide” ismeant a polypeptide or fragment thereof having at least about 85%, 90%,95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence providedbelow and that specifically binds a Trk receptor (e.g., TrkA, TrkC).JR-100 is the commercial name for sPDNF.

(SEQ ID NO: 5) 1 MGKTVVVASRMFWLMFFVPLLLAICPSEPAYALAPGSSRVELFKRKNSTVPFEDKAGKVT 61 ERVVHSFRLPALVNVDGVMVAIADARYDTSNDNSLIDTVAKYSVDDGETWETQIAIKNSR 121 VSSVSRVVDPTVIVKGNKLYVLVGSYYSSRSYWSSHGDARDWDILLAVGEVTKSTAGGKI 181 TASIKWGSPVSLKKFFPAEMEGMHTNQFLGGAGVAIVASNGNLVYPVQVTNKKKQVFSKI 241 FYSEDDGKTWKFGKGRSDFGCSEPVALEWEGKLIINTRVDWKRRLVYESSDMEKPWVEAV 301 GTVSRVWGPSPKSNQPGSQSSFTAVTIEGMRVMLFTHPLNFKGRWLRDRLNLWLTDNQRI 361 YNVGQVSIGDENSAYSSVLYKDDKLYCLHEINTDEVYSLVFARLVGELRIIKSVLRSWKN 421 WDSHLSSICTPADPAASSSESGCGPAVTTVGLVGFLSGNASQNVWEDAYRCVNASTANAE 481 RVRNGLKFAGVGGGALWPVSQQGQNQRYRFANHAFTLVASVTIHEAPRAASPLLGASLDS 541 SGGKKLLGLSYDEKHQWQPIYGSTPVTPTGSWETGKRYHLVL TMANKI

By “parasite-derived neurotrophic factor (PDNF) nucleic acid molecule”is meant a polynucleotide encoding a parasite-derived neurotrophicfactor polypeptide or fragment thereof. Exemplary PDNF nucleic acidsequences are provided in FIGS. 1A and 1C-1, 1C-2 and 1C-3.

By “soluble parasite-derived neurotrophic factor (sPDNF) nucleic acidmolecule” is meant a polynucleotide encoding a soluble parasite-derivedneurotrophic factor polypeptide or fragment thereof.

(SEQ ID NO: 6) 1 atggggaaaacagtcgttgtggccagtaggatgttctggctaatgtttttcgtgccgctt 61 cttcttgcgatctgccccagcgagcccgcgtacgccttggcacccggatcgagccgagtt 121 gagctgtttaagcgtaagaattcgacggtgccgtttgaagacaaggccggcaaagtcacc 181 gagcgggttgtccactcgttccgcctccccgcccttgttaatgtggacggggtgatggtt 241 gccatcgcggacgctcgctacgacacatccaatgacaactccctcattgatacggtggcg 301 aagtacagcgtggacgatggggagacgtgggagacccaaattgccatcaagaacagccgt 361 gtatcgtctgtttctcgtgtggtggatcccaccgtgattgtgaagggcaacaagctttac 421 gtcctggttggaagctactatagttcgagaagctactggtcgtcgcatggtgatgcgaga 481 gactgggatattctgcttgccgttggtgaggtcacgaagtccactgcgggcggcaagata 541 actgcgagtatcaaatgggggagccccgtgtcactgaagaagttttttccggcagaaatg 601 gaaggcatgcacacaaatcaatttcttggcggcgcgggtgttgccattgtagcgtccaac 661 gggaatcttgtgtaccctgtgcaggttacgaacaaaaagaagcaagttttctccaagatc 721 ttctactcggaagatgatggcaagacgtggaagtttgggaagggtaggagcgattttggc 781 tgctctgaacctgtggcccttgagtgggaggggaagctcatcataaacacccgagttgac 841 tggaaacgccgtctggtgtacgagtccagtgacatggagaaaccgtgggtggaggctgtc 901 ggaaccgtctcgcgtgtgtggggcccctcaccaaaatcgaaccagcccggcagtcagagc 961 agcttcactgccgtgaccatcgaaggaatgcgtgtgatgctcttcacacacccgctgaat 1021 tttaagggaaggtggctgcgcgaccgactgaacctctggctgacggataaccagcgcatt 1081 tataacgttgggcaagtatccattggtgatgaaaattccgcctacagctccgtcctgtac 1141 aaggatgataagctgtactgtttgcatgagatcaacacggacgaggtgtacagccttgtt 1201 tttgcacgcctggttggcgagctacggatcattaaatcagtgctgcggtcctggaagaat 1261 tgggacagccacctgtccagcatttgcacccctgctgatccagccgcttcgtcgtcagag 1321 agtggttgtggtcccgctgtcaccacggttggtcttgttggctttttgtccggcaacgcc 1381 tcccaaaacgtatgggaggatgcgtaccgctgcgtcaacgcaagcacggcaaatgcggag 1441 agggttcggaacggtttgaagtttgcgggggttggcggaggagcgctttggccggtgagc 1501 cagcaggggcagaatcagcggtatcgttttgcaaaccacgcgttcacgctggtggcgtcg 1561 gtgacgattcacgaggctccgagggccgcgagtcccttgctgggtgcgagcctggactct 1621 tctggcggcaaaaaactcctggggctctcgtacgacgagaagcaccagtggcagccaata 1681 tacggatcaacgccggtgacgccgacgggatcgtgggagacgggtaaaaggtaccacttg 1741 gttcttacgatggcgaataaaatt

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

By “reference” is meant a standard or control condition.

A “reference sequence” is a defined sequence used as a basis forsequence comparison.

By “small molecule” is meant any chemical compound.

By “specifically binds” is meant a compound or agent (e.g., PDNF) thatrecognizes and binds a polypeptide of the invention (e.g., TrkA, TrkC),but which does not substantially recognize and bind other molecules in asample.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Any compounds, compositions, or methods provided herein can be combinedwith one or more of any of the other compositions and methods providedherein.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural. Thus, for example, reference to “anamino acid substitution” includes reference to more than one amino acidsubstitution.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to.”

As used herein, the terms “comprises,” “comprising,” “containing,”“having” and the like can have the meaning ascribed to them in U.S.Patent law and can mean “includes,” “including,” and the like;“consisting essentially of or” consists essentially” likewise has themeaning ascribed in U.S. Patent law and the term is open-ended, allowingfor the presence of more than that which is recited so long as basic ornovel characteristics of that which is recited is not changed by thepresence of more than that which is recited, but excludes prior artembodiments.

Other features and advantages of the invention will be apparent from thefollowing description of the desirable embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C-1, 1C-2, 1C-3, and 1D provide the nucleotide and aminoacid sequences of exemplary PDNFs. FIG. 1A illustrates the nucleotidesequence of the PDNF gene, clone 19Y (SEQ ID NO: 1) deposited in GenBankunder accession number AJ002174, having an open-reading frame beginningat position 370. FIG. 1B illustrates the amino acid sequence of the PDNF(SEQ ID NO: 2) encoded by clone 19Y deposited in GenBank under accessionnumber AJ002174. FIGS. 1C-1, 1C-2 and 1C-3 illustrate the nucleotidesequence of the PDNF gene, clone 7F (SEQ ID NO: 3) deposited in GenBankunder accession number M61732, having an open-reading frame beginning atposition 484. FIG. 1D illustrates the amino acid sequence of the PDNF(SEQ ID NO: 4) encoded by clone 7F deposited in GenBank under accessionnumber M61732. The PDNF comprises a catalytic domain (amino acidresidues 33-666 of SEQ ID NO: 4), and a tandem repeat domain (amino acidresidues 667-1162 or SEQ ID NO: 4).

FIGS. 2A-2F show that intravenous administration of soluble PDNF (sPDNF)into naïve mice increased expression of cardiac and hepatic stem cellmarkers independent of TLR signaling, expanded cardiac Sca-1⁺ cells, andincreased expression of IL 1-Ra, TSG-6 and COX-2. FIG. 2A is a graphshowing stem cell marker expression in mice administered sPDNF. One setof three wild type C57BL/6 mice received intravenous (IV) PBS without(PBS) or with sPDNF (2 mg/kg) at 0, 3 and 24 hours (sPDNF 1d), and asecond set IV sPDNF (2 mg/kg) for 6 days (sPDNF 6d). Mice weresacrificed 3 hr post injection and the indicated stem cells markers werequantified in the ventricles by real time PCR, and normalized to HPRT;*, p<0.05, **, p<0.001, ***, p<0.0001; SCF, stem cell factor, ns, notsignificant. FIG. 2B is a graph showing stem cell marker expression inMyD88^(−/−) mice administered sPDNF. TLR-deficient MyD88^(−/−) mice(C57BL/6 background) (2/group) were injected IV with a single dose ofsPDNF (2 mg/kg), sacrificed at 3 hours post-injection, and the indicatedcardiac stem cell markers assessed by qPCR. Data were normalized to HPRTand made relative to vehicle-treated controls (set at 1.0 andrepresented by the dashed line); **, P<0.01; and ***, P<0.005. FIG. 2Cis a graph showing stem cell marker expression in mice administeredsPDNF at various time points after injection. C57BL/6 mice (5 per group)were injected IV with PBS or sPDNF (3 mg/kg body weight), and sacrificedat 0, 1, 3 and 7 hours later. Cardiac Sca-1 and SCF transcripts werequantified by qPCR; **, P<0.001; ***, P<0.0005. FIG. 2D is a graphshowing quantitation of Sca-1 expression in various organs of miceadministered sPDNF. C57BL/6 mice (3 per group), average of twoexperiments) were injected IV with sPDNF at 4 mg/kg body weight or PBS,sacrificed 3 hours later, and Sca-1 mRNA quantified by qPCR in theliver, heart, colon and bone marrow (*, P<0.05, **, P<0.001; ns, notsignificant; dotted line represents Sca-1 mRNA of vehicle-treated miceset at 1.0, data are representative of 2 experiments. FIG. 2E showimages of heart tissue samples with staining for Sca-1 from miceinjected with vehicle (left image) or PDNF (right image). C57BL/6 mice(2 per group) were injected IV three times (0, 3, and 24 h) with PBS (IVVehicle) or 2 mg/kg sPDNF (IV sPDNF), and sacrificed 48 hours after thefirst injection. Heart (ventricle) was fixed in paraformaldehyde, andtissue sections stained with a rabbit antibody against Sca-1 and DAPI.Normal rabbit IgG did not stain cardiac cells. Graph insert shows thepercentage of Sca-1⁺ cells per field determined by dividing the numberof Sca-1+ cells by the total number of cells (i.e., DAPI+ nuclei) perfield, in 12-14 random fields from 3 tissue sections per condition; ***,P<0.001; two experiments, similar results. FIG. 2F is a graph showingquantification of expression of inflammatory markers in miceadministered sPDNF. Wild type and naïve mice (5 per group) were injectedIV with a single dose of sPDNF (4 mg/kg), sacrificed 3 hrpost-injection, and the L-1Ra, TSG-6, COX-2, and IDO transcriptsquantified in the heart and liver. Data are combined from two separateexperiments; ***, P<0.001.

FIGS. 3A and 3B show that intravenous sPDNF triggered a dramaticreduction in inflammatory infiltrates in the heart of mice with chronicChagas cardiomyopathy (CCC). FIG. 3A depicts a graph showing in vivoexperimental design, groups, treatment and timeline for the infectiveand anti-inflammatory effects of sPDNF. C57BL/6 mice (7 per group) wereinjected intraperitoneally with T. cruzi Colombian strain (8×10² permouse) and, four months later, intravenously with PBS (vehicle) or sPDNF(1 mg/kg body weight) at 0, 3, and 24 hours, weekly for 3 weeks. Micewere sacrificed 50 days after the first set of IV sPDNF treatment. FIG.3B shows images of heart (ventricle) paraffin-embedded, sectioned, andstained with H&E. Arrows indicate foci of inflammatory infiltrates,which were abundant in sham-treated (IV PBS) CCC mice and rare in IVsPDNF-treated CCC mice. Similar histology was found in other tissuesections and in a distinct set of sPDNF-treated and PBS-sham mice.

FIGS. 4A-4C show that intravenous sPDNF reduced cardiac fibrosis in micewith chronic Chagas cardiomyopathy (CCC). The same mice and experimentalprotocol as described in FIG. 3A were used except that paraffin-embeddedheart sections were stained with Masson-Trichrome to visualize collagenin fibrotic areas (blue) and viable muscle fibers (red). FIGS. 4A and 4Bdisplay sections of atria of mice with CCC administered IV PBS or IVsPDNF, respectively. FIG. 4C is a graph showing the amount of cardiacfibrosis, as determined using ImageJ software (NIH) in four distinctsections of atria and ventricles. Relative cardiac fibrosis is definedas the ratio of blue pixels to total pixels (blue+red) normalized tocorresponding areas of the heart of uninfected mice set at 1.0 (dottedline); **, P<0.01, ns, not significant relative uninfected hearts.

FIGS. 5A-5F show that intravenous sPDNF triggered a global decrease inthe transcripts of cardiac inflammatory and fibrogenic markers, andincreased expression of the anti-inflammatory TSG-6 in the heart andliver of mice with chronic Chagas cardiomyopathy (CCC). FIG. 5A is agraph showing transcript expression for inflammatory cytokines in heartsof the infected mice, as assessed by qPCR for the indicated transcripts,which were normalized to HPRT and made relative to uninfectedvehicle-treated mice. Values for IFN-λ, were reduced by 3-fold; **,p<0.01. FIG. 5B is a graph showing transcript expression forinflammatory cell markers in hearts of the infected mice, as assessed byqPCR for the indicated transcripts, which were normalized to HPRT andmade relative to uninfected vehicle-treated mice. Values for CD8, andCD4 were reduced by 15-, and 1.5-fold, respectively; **, p<0.01, ***,p<0.001.

FIG. 5C is a graph showing transcript expression for inflammatorycytokines in hearts of the infected mice, as assessed by qPCR for theindicated transcripts, which were normalized to HPRT and made relativeto uninfected vehicle-treated mice. FIG. 5D is a graph showing percentreduction of cardiac inflammatory markers of mice chronically infectedwith T. cruzi, using data from FIGS. 5A-5C. Baseline represents meantranscripts of uninfected mice. FIG. 5E is a graph showing expression ofTSG-6 transcripts in heart and liver of T. cruzi infected mice. Heart(ventricles+atria) and liver of mice (5 per group) infected acutely(25-day post injection) (Acute) or chronically (6-months PI, CCC, sameas in FIGS. 3A and 3B) with T. cruzi Colombian were assessed for TSG-6transcript by qPCR and plotted relative to uninfected mice set at 1.0;**, P<0.001. Note that TSG-6 mRNA is increased in the heart and liver ofacute, but not chronic, infected, and that IV sPDNF in chronicallyinfected mice restored TSG-6 transcript to levels seen in acutelyinfected mice. FIG. 5F is a bar graph showing TSG-6 serum levelsassessed by ELISA (MyBiosource) in mice uninfected (Un-Inf) or bearingCCC and injected IV with vehicle PBS or sPDNF following the protocoldescribed in FIGS. 3A and 3B. Bars represent the average (±SD) of theemice per point; two separate experiments, **, P<0.01.

FIG. 6 illustrates a treatment scheme for treating IBD with sPDNF.sPDNF, at the dose of 1 mg/kg per injection, is administeredintraperitoneally at 0 hr, 3 hr, and 24 hr, one day per week for 3weeks, on day 10, day 17, and day 24, as indicated by the arrows.

FIGS. 7A-7D show that both T. cruzi and sPDNF increased expression ofMCP-1 and FKN transcripts in primary cultures of cardiomyocytes andcardiac fibroblasts. FIG. 7A is a graph depicting MCP-1 and FKNexpression in cardiomyocytes of mice infected with T. cruzi. FIG. 7B isa graph depicting MCP-1 and FKN expression in cardiac fibroblasts ofmice infected with T. cruzi. T. cruzi infection: Primary cultures ofmouse cardiomyocytes (FIG. 7A) and cardiac fibroblasts (FIG. 7B) wereplated in 6-well plates, infected with T. cruzi Tulahuen strain (MOI=10)for 3, 24, and 72 hr, and MCP-1 and FKN transcripts were quantified byqPCR. Chemokine gene expression was normalized to HPRT and fold changecalculated relative to uninfected cells. Points represent mean±SD foldchange expression of triplicate samples. The experiment was repeated,and similar results were obtained. FIG. 7C is a graph depicting MCP-1and FKN expression in cardiomyocytes of mice injected with sPDNF. FIG.7D is a graph depicting MCP-1 and FKN expression in cardiac fibroblastsof mice injected with sPDNF. sPDNF stimulation: Primary cardiomyocytes(FIG. 7C) and cardiac fibroblasts (FIG. 7D) were plated in 6-wellplates, serum starved overnight, then stimulated with the indicatedconcentration of sPDNF (3 hr), and MCP-1 and FKN transcripts werequantified by qPCR. Chemokine gene expression was normalized to HPRT andfold change calculated relative to vehicle-treated cells; *P<0.05,**P<0.01, and ***P<0.005.

FIGS. 8A-8C show that sPDNF increased MCP-1 and FKN expression in H9c2cardiomyocytes dose-dependently and in a pulse-like manner. FIG. 8A is agraph showing dose-response of MCP-1 secretion to sPDNF stimulation.H9c2 cardiomyocytes were plated in 6-well plates, grown in low serum(0.1% FCS) overnight, and stimulated with the indicated doses of sPDNFfor 3 hr. Secretion of MCP-1, MIP-2α and IL-6 was determined by ELISA.FIG. 8B is a graph showing dose-response of MCP-transcript expression.H9c2 cardiomyocytes were plated in 6-well plates, grown in low serum(0.1%) overnight, and stimulated with the indicated doses of sPDNF for 3hr, and MCP-transcript quantified by qPCR. Chemokine gene expression wasnormalized to HPRT and fold change calculated relative tovehicle-treated cells. Points represent mean±SD fold change expressionof triplicate samples; *P<0.05, **P<0.01, and ***P<0.005. FIG. 8C is agraph showing time course of MCP-1 and FKN transcripts expression. H9c2cardiomyocytes were plated in 6-well plates (triplicate), grown in lowserum (0.1%) overnight, stimulated with sPDNF (1 μg/ml) for theindicated times, and MCP-1 and FKN (data not shown) transcriptsquantified by qPCR. Three experiments gave similar results.

FIGS. 9A-9E show that neurotrophic receptors TrkA and TrkC were targetedby sPDNF for increased expression of MCP-1 and FKN in cardiomyocytes.FIG. 9A is a graph showing the effect of the Trk antagonist, K252a, onMCP-1 transcript expression in cardiomyocytes treated with sPDNF. FIG.9B is a graph showing the effect of the Trk antagonist, K252a, on FKNtranscript expression in cardiomyocytes treated with sPDNF.Pharmacological inhibition: H9c2 cardiomyocytes were stimulated withsPDNF (1 μg/ml, 3 hr) without pretreatment (sPDNF) or after pretreatmentwith K252a (200 nM, 1 hr) (K252a+sPDNF), and transcripts of MCP-1 (FIG.9A) and FKN (FIG. 9B) quantified by qPCR. Chemokine gene expression wasnormalized to HPRT and fold change calculated relative tovehicle-treated cells. Bars represent mean±SD fold change expression ofduplicate samples, *P<0.05 and **P<0.01; experiment performed threetimes, each giving similar results. FIG. 9C is a graph showing theeffect of Trk neutralizing antibodies on MCP-1 transcript expression incardiomyocytes treated with sPDNF. FIG. 9D is a graph showing the effectof Trk neutralizing antibodies on FKN transcript expression incardiomyocytes treated with sPDNF. Neutralizing antibodies: H9c2cardiomyocytes were stimulated with sPDNF (1 μg/ml, 3 hr) withoutpretreatment (+sPDNF) or after pretreatment with neutralizing antibodies(1 μg/ml, 30 min) against TrkA (α-TrkA+sPDNF), TrkB (α-TrkB+sPDNF), andTrkC (α-TrkC+sPDNF) followed by sPDNF), and transcripts of MCP-1 (FIG.9C) and FKN (FIG. 9D) quantified by qPCR. Chemokine gene expression wasnormalized to HPRT and fold change calculated relative tovehicle-treated cells. Bars represent mean±SD fold change expression ofduplicate samples, *P<0.05 and **P<0.01, ns=not significant. FIG. 9E isa graph showing the effect of TrkA and TrkC RNA silencing on MCP-1secretion in cardiomyocytes treated with sPDNF. RNA silencing: H9c2cardiomyocytes were transfected with lentiviral particles containingshRNAs targeting GFP (control), TrkA (clones 1-4) and TrkC (clones 1-5).After 7-8 d, cells were serum starved (2 hr) and stimulated with vehicleor sPDNF (4 μg/ml, 3 hr), and the concentration of MCP-1 secreted in thesupernatants was determined by ELISA; ns=not significant, *P<0.05,**P<0.01, and ***P<0.005 (relative to shGFP transfected cells).

FIGS. 10A and 10B show that MCP-1 and FKN mRNAs increased in acutechagasic hearts in proportion to parasite burden. FIG. 10A is a graphshowing the kinetics of cardiac MCP-1 mRNA in response to acute T. cruziinfection in relation to heart parasite burden. FIG. 10B is a graphshowing the kinetics of cardiac MCP-1 and FKN mRNA in response to acuteT. cruzi infection in relation to heart parasite burden. Groups ofC57BL/6 mice (3 per group) were subcutaneously infected with T. cruzi,and, at the indicated timepoints, mice were sacrificed and MCP-1, FKNand parasite burden quantified in their heart (ventricles+atria) byqPCR.

FIGS. 11A-11H show that intravenous sPDNF increased expression of MCP-1and FKN in the heart and other organs of wild type and MyD88^(−/−) mice.Wild type C57BL/6 mice were used in the results depicted in FIGS.11A-11D. FIG. 11A is a graph depicting that a single dose of intravenous(IV) sPDNF increased MCP-1 and FKN transcripts in the heart in apulse-like response. Groups of mice (2 per group) were injected withvehicle (Veh) or sPDNF (150 μg/mouse) and sacrificed at 3, 6, 9 and 12hr post-injection, and cardiac MCP-1 and FKN transcripts were quantifiedby qPCR. Points represent fold change of the mean±SD relative to Vehinjected mice; *P<0.05 and **P<0.01. Experiment repeated three timeswith similar results; liver also responded with similar pulse likekinetics. FIG. 11B is a graph depicting that organ distribution of MCP-1increased following a single IV dose of sPDNF. Experimental designanalogous to that summarized above. FIG. 11C is a graph showing that asingle IV dose of sPDNF augments MCP-1 and FKN transcriptsdose-dependently in the heart. FIG. 11D is a graph showing that a singleIV dose of sPDNF augments MCP-1 and FKN transcripts dose-dependently inthe liver. For the results depicted in FIGS. 11C and 11D, groups ofthree mice were injected with various doses of sPDNF, and heart andliver harvested 3 hr post-injection to quantify MCP-1 and FKN by qPCR.The results are plotted as a composite of three distinct experiments.The increase in hepatic FKN mRNA was not statistically significant; ***P<0.005. For the results depicted in FIGS. 11E-11H, TLR-deficientMyD88^(−/−) mice (two per group) were injected IV with vehicle (PBS) orsPDNF (100 μg per mouse), sacrificed 3 hr post-injection. FIG. 11E is agraph showing MCP-1 expression in the heart of MyD88^(−/−) mice injectedwith sPDNF. FIG. 11F is a graph showing FKN expression in the heart ofMyD88^(−/−) mice injected with sPDNF. FIG. 11G is a graph showing MCP-1expression in the liver of MyD88^(−/−) mice injected with sPDNF. FIG.11H is a graph showing FKN expression in the liver of MyD88^(−/−) miceinjected with sPDNF.

FIGS. 12A-12D show that multiple intravenous injections of sPDNFincreased transcripts of MCP-1 and FKN receptors CCR2 and CX3CR1 in theheart and liver. For the results depicted in FIGS. 12A and 12B, mice(3/group) were injected with various single doses of IV sPDNF, andsacrificed 3 hr post-injection. FIG. 12A is a graph showing the mRNAexpression of MCP-1 receptor CCR2, as quantified by qPCR in the heartand liver. FIG. 12B is a graph showing the mRNA expression of FKNreceptor CX3CR1, as quantified by qPCR in the heart and liver. Note thatreceptor transcripts did not increase following a single dose of IVsPDNF at 3 hr post-injection and 24 hr post-injection. For the resultsdepicted in FIGS. 12C and 12D, mice (3/group) were injected with vehicle(Veh) or multiple doses of IV sPDNF (25 μg per injection) at 0, 3 and 24hr, and sacrificed 24 hr after the last injection. FIG. 12C is a graphshowing mRNA expression of CCR2, as quantified by qPCR in the liver,colon and heart (ventricles and atria). FIG. 12D is a graph showing mRNAexpression of CX3CR1, as quantified by qPCR in the liver, colon andheart (ventricles and atria). Notice a statistically significantupregulation of MCP-1 and FKN receptors in the heart and liver 48-hafter the start of three doses of IV sPDNF spaced within a 24 h span;*P<0.05, and ** P<0.01

DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions and methods for treatinginflammatory diseases or disorders, such as cardiac or hepaticinflammatory diseases, using parasite-derived neurotrophic factor(PDNF), or a fragment of PDNF that binds to the TrkA and/or TrkCReceptor. The compositions and methods are used to treat, for example,myocarditis, cardiomyopathy, endocarditis, pericarditis, Chagas disease,or hepatitis. The methods may also be used to treat an inflammatorydisease of the gastrointestinal (GI) tract, pancreas, or a liverdisease.

As reported in detail below, the invention is based in part on thediscovery that PDNF can promote the regeneration of cardiac tissue, aswell as reduce the inflammation of cardiac tissue. In particular, asdisclosed and exemplified herein, the inventor discovered thatadministration of a PDNF polypeptide promoted both the proliferation andthe mobilization of stem cells to non-neuronal tissues (e.g., heart,liver, pancreas, gastrointestinal tract, etc.).

Parasite-Derived Neurotrophic Factor (PDNF)

The human parasite Trypanosoma cruzi, the agent of Chagas disease,expresses a membrane-bound neuraminidase, also known as trans-sialidase(TS), or parasite-derived neurotrophic factor (PDNF) because it bindsand activates nerve growth factor (NGF) receptor TrkA in neuronal cells.

PDNF is an enzyme expressed on the T. cruzi's surface and catalyzes thetransfer of sialic acid from host glycoconjugates to glycoproteinmolecules on the surface of the parasite. See, Schenkman et al., Exp.Parasitol., 72:76 86 (1991). The enzyme is present both in theepimastigote form (i.e., in the invertebrate vector) and in thetrypomastigote form (i.e., infectious form that circulates in the bloodof the vertebrate host). See, Agusti et al., Glycobiology, 7(6):731 5,(1997).

FIGS. 1A-1D show the nucleotide sequences and amino acid sequences oftwo naturally-occurring PDNFs. FIG. 1A illustrates the nucleotidesequence of the T. cruzi PDNF gene, clone 19Y (SEQ ID NO: 1), depositedin GenBank under accession number AJ002174, having an open-reading framebeginning at position 370. FIG. 1B illustrates the amino acid sequenceof the T. cruzi PDNF (SEQ ID NO: 2) encoded by clone 19Y deposited inGenBank under accession number AJ002174. FIGS. 1C-1, 1C-2 and 1C-3illustrate the nucleotide sequence of the T. cruzi PDNF gene, clone 7F(SEQ ID NO: 3) deposited in GenBank under accession number M61732,having an open-reading frame beginning at position 484. FIG. 1Dillustrates the amino acid sequence of the T. cruzi PDNF (SEQ ID NO: 4)encoded by clone 7F deposited in GenBank under accession number M61732.The entire teachings of the information deposited in GenBank underaccession numbers AJ002174 and M61732 are incorporated herein byreference.

The naturally-occurring, full-length PDNF from T. cruzi trypomastigoteshas 4 distinct amino acid regions: (1) a N-terminal region withapproximately 380 amino acids, which shares about 30% sequence identityto bacterial sialidases; (2) a region with approximately 150 residuesthat does not show any similarity with any known sequence; (3) a regionwith homology to type III fibronectin (FnIII); and (4) a C-terminalregion containing 12 repeated amino acids, which is the immuno-dominantportion and which is required for enzyme oligomerization. The N-terminaland the FnIII regions are important for trans-sialidase activity.

The catalytic portion of a native trans-sialidase has two kinds ofenzymatic activities: (1) neuraminidase activity, which releases sialicacid from the complex carbohydrates; and (2) sialil-transferaseactivity, which catalyzes the transfer of sialic acid from glyconjugatedonors to terminal β-D galactose containing acceptors. See, Scudder etal., J. Biol. Chem., 268(13):9886 91 (1993). Residues 33-666 of SEQ IDNO: 2 correspond to the catalytic domain of clone 19Y. Amino acidresidues 1 to 596 of SEQ ID NO: 4 encompass the catalytic domain ofclone 7F.

The full-length native trans-sialidase also has a long 12-amino acidtandem repeat domain in the C-terminus, previously identified as SAPA(i.e., Shed-Acute-Phase-Antigens). Although the tandem repeat is notdirectly involved in the catalytic activity, it stabilizes thetrans-sialidase activity in the blood to increase the half-life of theenzyme from about 7 to about 35 hours. See, Pollevick et al., Mol.Biochem. Parasitol. 47:247 250 (1991) and Buscaglia et al., J. Infect.Dis., 177(2):431 6 (1998). Amino acid residues 667-1162 of SEQ ID NO: 4correspond to the C-terminal tandem repeat of clone 7F. The C-terminaltandem repeat domain is not required for the neurotrophic activity ofthe PDNF. See, e.g., Chuenkova et al., U.S. Application Publication Nos.2009/0117593 and 2006/0229247.

PDNF binds and activates the NGF receptor TrkA to trigger PI3K/Akt andMAPK/Erk signaling (Chuenkova and PereiraPerrin J. Neurochem.,91:385-394 (2004); Woronowicz et al., Glycobiology 14:987-98 (2004)).However, neurotrophic receptor activation is independent of sialic acidbinding because the neuraminidase/trans-sialidase 1) does not lose Trkreceptor recognition after point mutations that sharply reduceneuraminidase/trans-sialidase activities (11), 2) Trk-binding activityis mimicked by a 24-mer synthetic peptide (13), and 3) binds andactivates Trk receptors in sialic acid-deficient cells (60).Furthermore, the enzyme, after phosphorylation by Akt kinase, promotescell survival in the cytosol where sialyl-conjugate substrates areabsent, (14). Neuraminidase/trans-sialidase is called PDNF to underscoreparacrine growth factor function independent of sialic acid binding.Binding to TrkC leads to T cruzi entry into cardiomyocytes and cardiacfibroblasts, whereas binding to TrkA triggers cardioprotection byautocrine and paracrine mechanisms (4, 5). A peptide mapping approachhas revealed that a bacterially-expressed truncated PDNF correspondingto amino acids 1-445 of SEQ ID NO:2 promoted survival anddifferentiation of TrkA-positive PC12 cells while truncated PDNFequivalent to amino acids 1-425 of SEQ ID NO:2 did not (Chuenkova andPereira, Mol. Biol. Cell 11:1487-1498 (2000)). This finding indicatesthat the amino acid sequence corresponding to 425-445 of SEQ ID NO: 2 isinvolved in the TrkA-mediated signaling. A predicted three-dimensionalstructure of the catalytic domain of PDNF is provided in FIG. 34 of U.S.Application Publication No. 2009/0117593.

The invention also encompasses the use of a PDNF fragment that comprisesa portion, but not the full-length sequence of PDNF, while retaining theTrkA-binding activity or the ability to activate TrkA signaling pathway.The PDNF fragments of the invention may or may not have neuraminidase ortrans-sialidase catalytic activity as desired. Residues 33-666 of SEQ IDNO: 2 is an example of a fragment of PDNF that retains the TrkA-bindingactivity. Residues 425-455 of SEQ ID NO: 2 is another example of afragment of PDNF that retains the TrkA-binding activity. In anotherexemplary embodiment, the PDNF fragment is residues 1 to 588 of SEQ IDNO: 4. In another exemplary embodiment, the PDNF fragment is residues 1to 596 of SEQ ID NO: 4.

The PDNF polypeptide or PDNF fragment of the invention can be anaturally occurring protein that has TrkA-binding activity (e.g., bindsto TrkA receptor or activates TrkA signaling pathway), or an activevariant of a naturally occurring protein.

As used herein, “active variants” refers to variant peptides whichretain TrkA-binding activity (e.g., binds to TrkA receptor or activatesTrkA signaling pathway). An “active variant” may or may not have to haveneuraminidase or trans-sialidase catalytic activity as desired. Anactive variant differs in amino acid sequence from a reference PDNF(such as the PDNF encoded by clone 19Y deposited in GenBank underaccession number AJ002174 (SEQ ID NO: 2), or the PDNF encoded by clone7F deposited in GenBank under accession number M61732 (SEQ ID NO: 4)),or a reference PDNF fragment, but retains TrkA-binding activity (e.g.,retains the ability to bind to TrkA receptor or activates TrkA signalingpathway).

Generally, differences are limited so that the sequences of thereference polypeptide and the active variant are closely similar overalland, in many regions, identical. An active variant of PDNF or PDNFfragment and a reference PDNF or PDNF fragment can differ in amino acidsequence by one or more amino acid substitutions, additions, deletions,truncations, fusions or any combination thereof. Preferably, amino acidsubstitutions are conservative substitutions. A conservative amino acidsubstitution refers to the replacement of a first amino acid by a secondamino acid that has chemical and/or physical properties (e.g., charge,structure, polarity, hydrophobicity/hydrophilicity) which are similar tothose of the first amino acid. Conservative substitutions includereplacement of one amino acid by another within the following groups:lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate(E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine(Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine(I), proline (P), phenylalanine (F), tryptophan (W), methionine (M),cysteine (C) and glycine (G); F, W and Y; C, S and T.

Preferred size of PDNF fragments (including active variants of a PDNFfragment) is at least 10 amino acids (a.a.), at least 12 a.a., at least15 a.a., or at least 20 a.a. As described herein, such fragments (andactive variants of the PDNF fragment) bind to TrkA receptor.

Active variants of PDNF or PDNF fragments include naturally occurringvariants (e.g., allelic forms) and variants which are not known to occurnaturally.

In one embodiment, an active variant of PDNF shares at least about 85%amino acid sequence similarity or identity with a naturally occurringPDNF (e.g., SEQ ID NO: 2, SEQ ID NO:4), preferably at least about 90%amino acid sequence similarity or identity, and more preferably at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% amino acid sequence similarity or identity withsaid PDNF. Preferably, the percentage of identity is calculated over thefull length of the active variant.

In certain embodiments, the active variant comprises fewer amino acidresidues than a naturally occurring PDNF. In this situation, the variantcan share at least about 85% amino acid sequence similarity or identitywith a corresponding portion of a naturally occurring PDNF (e.g., aminoacid residues 33-666 of SEQ ID NO: 2), preferably at least about 90%amino acid sequence similarity or identity, and more preferably at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% amino acid sequence similarity or identity with acorresponding portion of said PDNF.

Portions of the amino acid sequence of PDNF which correspond to avariant and amino acid sequence similarity or identity can be identifiedusing a suitable sequence alignment algorithm, such as ClustalW2(http://www.ebi.ac.uk/Tools/clustalw2/index.html) or “BLAST 2 Sequences”using default parameters (Tatusova, T. et al., FEMS Microbiol. Lett.,174:187-188 (1999)).

Active variants of PDNF or PDNF fragments can be prepared using suitablemethods, for example, by direct synthesis, mutagenesis (e.g., sitedirected mutagenesis, scanning mutagenesis) and other methods ofrecombinant DNA technology. Active variants can be identified and/orselected using a suitable assay, such as the co-immunoprecipitation andkinase assays. For example, TrkA binding activities can be shown byco-immunoprecipitation (or crosslinking) of TrkA receptor and the PDNFfragment or variant. Activation of TrkA pathway can be shown by kinaseassay, such as Ras/MAPK kinase assay or Akt kinase assay. See e.g., US2009/0117593 A1.

Fusion proteins comprising PDNF or a fragment of PDNF are alsocontemplated. A fusion protein may encompass a polypeptide comprisingPDNF (e.g., SEQ ID NO: 2, SEQ ID NO:4), a PDNF fragment (e.g., aminoacid residues 33-666 of SEQ ID NO: 2) or an active variant thereof as afirst moiety, linked via a covalent bond (e.g., a peptide bond) to asecond moiety (a fusion partner) not occurring in PDNF as found innature. Thus, the second moiety can be an amino acid, oligopeptide orpolypeptide. The second moiety can be linked to the first moiety at asuitable position, for example, the N-terminus, the C-terminus orinternally. In one embodiment, the fusion protein comprises an affinityligand (e.g., an enzyme, an antigen, epitope tag, a binding domain) anda linker sequence as the second moiety, and PDNF or a PDNF fragment asthe first moiety. Additional (e.g., third, fourth) moieties can bepresent as appropriate. The second (and additional moieties) can be anyamino acid, oligopeptide or polypeptide that does not interfere with theTrkA binding activity (e.g., the ability to bind to TrkA receptor oractivates TrkA signaling pathway) of PDNF. Fusion proteins can beprepared using suitable methods, for example, by direct synthesis,recombinant DNA technology, etc.

In certain embodiment, the fusion protein comprises a first moiety whichshares at least about 85% sequence similarity or identity with PDNF(e.g., SEQ ID NO:2, SEQ ID NO:4) or a fragment of PDNF (e.g., amino acidresidues 33-666 of SEQ ID NO:2), preferably at least about 90% sequencesimilarity or identity, and more preferably at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%sequence similarity or identity with the PDNF or PDNF fragment.Preferably, the percentage of identity is calculated over the fulllength of the first moiety.

Chagas Disease and Immune Response

Chagas disease, a major cause of morbidity and mortality in LatinAmerica (25, 27, 41), is commonly transmitted by blood-sucking reduviidbugs, which, during a blood meal, release Trypanosoma cruzi that, asmetacyclic trypomastigotes, gain access into the body through mucosalsurfaces or discontinuity in the skin, where they elicit a pronouncedinflammatory response. In the conjunctiva, inflammation and edema maylast many weeks and, in endemic areas, the unilateral eye inflammationserves as a diagnostic tool (Romana' sign). After a few days in thetransmission site, T. cruzi spreads throughout the body and, in theheart, triggers severe inflammation and tissue damage that often givesrise to ventricular repolarization, ejection defects, pleural effusion,and cardiomegaly (6, 35, 39). This response wanes in most (>95%)infected patients, who progress to symptomless and pathology-freeindeterminate phase that can last years or a lifetime. However, <5%acute chagasic patients progress to widespread and fulminatingmyocarditis.

Host survival and T. cruzi growth are controlled by innate and acquiredimmunity (25, 27, 41). During the initial stage of infection, Toll-likereceptor (TLR) family of pattern recognition proteins play an importantrole in T. cruzi recognition, as they are activated by parasitemolecules such as DNA, RNA or glycosylphosphatidyl-inositol (GPI)anchors, initiating a signaling cascade dependent on the adaptormolecule myeloid differentiation factor 88 (MyD88) that mediatesupregulation of pro-inflammatory genes pivotal to the resistance to T.cruzi infection. In addition, T. cruzi activates Nodi and inflammasome(NLRP3) pro-inflammatory pathways, and the end-result is an increase ininterleukin-1p (IL-1 p) (20), IL-6 (20, 55), IL-12 (1-3, 8, 29), TNFa(3, 7, 8, 29), interferon-p and interferon-Y (3, 9, 20, 24, 29, 43), andmonocyte chemoattractant protein-1 (MCP-1)/CCL2 and other chemokinesessential to the recruitment of inflammatory cells to infection sites(16, 54).

A most-studied mechanism underlying innate immunity in T. cruziinfection is Toll-like receptor (TLR) activation by lipids and otherparasite molecules. However, yet-to-be identified pathways should exist.It was reported that connective tissue growth factor (CTGF), also knownas CCN2, a matricellular protein implicated in fibrosis and inflammationin several disorders such as diabetic neuropathy (19), augmentsexpression of various pro-inflammatory cytokines, including thechemokine MCP-1, following binding and activation of neurotrophicreceptor TrkA on cardiomyocytes (57, 58). Given that CTGF/CCN2 receptoris TrkA, the same that sPDNF interacts with to promote downstreamsignaling and cell survival in host cells, including cardiac cells (4,5), the connection between T. cruzi PDNF activation of TrkA and/or TrkCon cardiomyocytes and cardiac fibroblasts and increased expression ofmediators of innate immunity was investigated.

As described herein, T. cruzi strongly increased expression of monocytechemoattractant protein-1 (MCP-1)/CCL2 and fractalkine (FKN)/CX3CL1 incellular and mouse models of heart infection. Mechanistically, increasedexpression of MCP-1 and FKN stemmed from the interaction ofparasite-derived neurotrophic factor (PDNF)/trans-sialidase withneurotrophic receptors TrkA and TrkC, as assessed by pharmacologicalinhibition, neutralizing antibodies and gene silencing studies. A singledose of intravenous PDNF into naïve mice resulted in a dose-dependentincrease in MCP-1 and FKN in the heart and liver, and pulse-likekinetics that peaked 3 hr post-injection. Intravenous PDNF alsoaugmented MCP-1 and FKN in TLR-deficient MyD88 knockout mice. Withoutbeing bound to a particular theory, this shows that PDNF has aTLR-independent action. Although single PDNF injections did not increaseMCP-1 and FKN receptors, multiple PDNF injections at short intervalsincreased expression of receptor transcripts in the heart and liver.Without being bound to a particular theory, this indicates thatsustained PDNF expression triggered cell recruitment at infection sites.Thus, given that MCP-1 and FKN are chemokines important to therecruitment of immune cells to combat inflammation triggers and toenhance tissue repair, these results describe a new mechanism in innateimmunity of T. cruzi infection mediated by Trk signaling. This pathwayhas similarities with an endogenous inflammatory and fibrotic pathwaythat results from cardiomyocyte-TrkA recognition by matricellularconnective tissue growth factor (CTGF/CCN2).

Use of PDNF for Treating Inflammatory Diseases

In one aspect, the invention provides a method of decreasinginflammation in a non-neuronal tissue of a subject, involvingadministering to the subject soluble parasite-derived neurotrophicfactor (sPDNF), or fragment thereof, in an amount effective to decreaseinflammation in the non-neuronal tissue.

In another aspect, the invention provides a method of decreasinginflammation in a cardiac, liver, pancreas, or gastrointestinal tissueof a subject, involving administering to the subject solubleparasite-derived neurotrophic factor (sPDNF), or a fragment thereof, inan amount effective to decrease inflammation in the cardiac, liver,pancreatic, or gastrointestinal tissue.

In still another aspect, the invention provides a method of increasingexpression of an anti-inflammatory factor in a non-neuronal cell ortissue, involving contacting the non-neuronal cell or tissue withsoluble parasite-derived neurotrophic factor (sPDNF), or a fragmentthereof, in an amount effective to increase expression of ananti-inflammatory factor in the non-neuronal cell or tissue.

In another aspect, the invention provides a method of treating a cardiacinflammatory disease in a subject in need thereof, involving:administering to the subject an anti-inflammatory or therapeuticallyeffective amount of parasite-derived neurotrophic factor (PDNF), or afragment of PDNF. In certain embodiments, the fragment of PDNF binds toTrkA Receptor. In certain embodiments, the subject does not have Chagasdisease. In certain embodiments, the subject does not have a mycoplasmainfection. In certain embodiments, the subject does not have a loss ofhematopoietic regeneration capacity. In certain embodiments, the cardiacinflammatory disease is myocarditis, cardiomyopathy, endocarditis, orpericarditis.

In one aspect, the invention provides a method of increasing stem cellnumber, involving contacting a non-neuronal cell or tissue with solubleparasite-derived neurotrophic factor (sPDNF), or fragment thereof.

In another aspect, the invention provides a method of increasing stemcell mobilization, involving contacting a non-neuronal cell or tissuewith soluble parasite-derived neurotrophic factor (sPDNF), or fragmentthereof.

In yet another aspect, the invention provides a method of increasingstem cell proliferation, involving contacting a non-neuronal cell ortissue with soluble parasite-derived neurotrophic factor (sPDNF), orfragment thereof.

In another aspect, the invention provides a method of promoting theproliferation or mobilization of cardiac stem cells in a subject in needthereof, involving: administering to the subject a therapeuticallyeffective amount of parasite-derived neurotrophic factor (PDNF), or afragment of PDNF. In certain embodiments, the fragment of PDNF binds toTrkA Receptor. In certain embodiments, the subject does not have Chagasdisease. In certain embodiments, the subject does not have a mycoplasmainfection. In certain embodiments, the subject does not have a loss ofhematopoietic regeneration capacity. In certain embodiments, the subjectis suffering from, or susceptible to myocarditis, cardiomyopathy,endocarditis, or pericarditis.

The PDNF or a TrkA binding fragment thereof described herein may also beused to treat fibrosis, in particular cardiac fibrosis. Cardiac fibrosisrefers to an abnormal thickening of the cardiac muscle or heart valvesdue to inappropriate proliferation of cardiac fibroblasts, with commoncharacteristics such as excessive collagen accumulation and defectivefunction caused by the replacement of normal tissues by fibrous tissues.Acute cardiac fibrosis includes responses to acute hypertension, trauma,infection, surgery, burn, radiation and chemotherapeutic agents. Chronicfibrosis is caused by other chronic diseases inducing chronichypertension, virus infection, diabetes, obesity, fatty liver anddermatosclerosis.

In one aspect, the invention provides a method of treating a hepaticinflammatory disease in a subject in need thereof, involving:administering to the subject an anti-inflammatory or therapeuticallyeffective amount of parasite-derived neurotrophic factor (PDNF), or afragment of PDNF. In certain embodiments, the fragment of PDNF binds toTrkA Receptor. In certain embodiments, the subject does not have Chagasdisease. In certain embodiments, the subject does not have a mycoplasmainfection. In certain embodiments, the subject does not have a loss ofhematopoietic regeneration capacity. In certain embodiments, the hepaticinflammatory disease is hepatitis or hepatic fibrosis.

In another aspect, the invention provides a method of promoting theproliferation or mobilization of hepatic progenitor cells in a subjectin need thereof, involving: administering to the subject atherapeutically effective amount of parasite-derived neurotrophic factor(PDNF), or a fragment of PDNF. In certain embodiments, the fragment ofPDNF binds to TrkA Receptor. In certain embodiments, the subject doesnot have Chagas disease. In certain embodiments, the subject does nothave a mycoplasma infection. In certain embodiments, the subject doesnot have a loss of hematopoietic regeneration capacity. In certainembodiments, the subject is suffering from, or susceptible to hepatitisor hepatic fibrosis.

In another aspect, the invention provides a method of treating aninflammatory disease of the gastrointestinal (GI) tract in a subject inneed thereof, involving: administering to the subject ananti-inflammatory or therapeutically effective amount ofparasite-derived neurotrophic factor (PDNF), or a fragment of PDNF,where the fragment of PDNF binds to TrkA Receptor. In certainembodiments, the subject does not have Chagas disease. Examples ofinflammatory diseases of the gastrointestinal (GI) tract include, forexample, inflammatory bowel diseases (IBD, with ulcerative colitis andCrohn's disease being the two major types of IBD), irritable bowelsyndrome, ileitis, chronic inflammatory intestinal disease, celiacdisease, etc.

In one aspect, the invention provides a method of treating a pancreaticinflammatory disease in a subject in need thereof, involving:administering to the subject an anti-inflammatory or therapeuticallyeffective amount of parasite-derived neurotrophic factor (PDNF), or afragment of PDNF. In certain embodiments, the fragment of PDNF binds toTrkA Receptor. In certain embodiments, the subject does not have Chagasdisease. In certain embodiments, the subject does not have a mycoplasmainfection. In certain embodiments, the subject does not have a loss ofhematopoietic regeneration capacity. In certain embodiments, the hepaticinflammatory disease is type 1 or type 2 diabetes.

In another aspect, the invention provides a method of treating a liverdisease in a subject in need thereof, involving: administering to thesubject an anti-inflammatory or therapeutically effective amount ofparasite-derived neurotrophic factor (PDNF), or a fragment of PDNF,where the fragment of PDNF binds to TrkA Receptor. In certainembodiments, the subject does not have Chagas disease. Examples of liverdiseases include, e.g., liver failure, hepatitis (acute or chronic),liver cirrhosis, toxic liver damage (for example alcohol), hepaticencephalopathy, hepatic coma, hepatic necrosis, etc. Other types ofdisease that can be treated by PDNF or PDNF fragments include, forexample, amyotrophic lateral sclerosis.

As disclosed and exemplified herein, PDNF, or a TrkA-binding fragmentthereof, is a stem cell renewal factor that promotes the proliferationand mobilization of stem cells such as cardiac stem cells and hepaticprogenitor cells, and can be used to treat cardiac or hepaticinflammatory diseases, in particular diseases characterized byinflammation and damage of cardiac or hepatic tissues.

As disclosed and exemplified herein, the inventor discovered that PDNFis a paracrine factor that promote both the proliferation andmobilization of stem cells. Although not wishing to be bound by aparticular theory, it is believed that PDNF can increase the number ofendogenous resident cardiac stem cells by at least two mechanisms.First, it is believed that PDNF promotes the proliferation of cardiacstem cells (as shown by the increased expression of cardiac stem cellmarkers Sca-1 and c-Kit) by binding to TrkA receptor and activating theTrkA-mediated kinas pathway. Second, it is also believed that PDNF canpromote the mobilization of cardiac stem cells by increasing thesynthesis of chemotatic factors (e.g., an increased expression ofchemokine CCL2 and its receptor CCR2).

Although not wishing to be bound by a particular theory, it is believedthat PDNF triggers the proliferation and mobilization of cardiac stemcells directly by activating TrkA receptor, as well as indirectly byincreasing the expression of nerve growth factor (NGF) (˜80-foldincrease in cardiac fibroblasts in some of the experiments). NGF canprotect against myocardial infarction partly by upregulating cardiacc-Kit⁺ stem cells. Hence, it is believed that PDNF promotes theproliferation and mobilization of cardiac progenitor cells both directly(via TrkA receptor) and indirectly (via increased expression of NGF).

The increase of cardiac stem cells, triggered by PDNF, results in anincreased regenerative activity (repair of damaged tissue), as stemcells can differentiate into mature cardiomyocytes. The increased stemcells also provide strong anti-inflammatory effect. In a model ofchronic Chagas disease, an intravenously-administered PDNF fragmentreduced cardiac inflammation (as reflected by CD8 lymphocyteinfiltrates, and pro-inflammatory cytokines such as tumor necrosisfactor-a), and nearly eliminated fibrosis. Such PDNF-triggered globalanti-inflammatory activity is attributed to the increased proliferationand mobilization stem cells, which can reduce or suppress inflammation.

The methods described herein use PDNF, or a TrkA-binding fragmentthereof, to trigger the expansion of endogenous cardiac stem cells,which provides certain advantages over stem cell therapy usingnon-cardiac progenitor cells. Prior stem cell clinical trials usingnon-cardiac progenitor cells has failed, with problems such asdifferentiation of the stem cells into non-cardiac cells instead ofcardiomyocytes, inefficient delivery (˜10% cardiac retention), cellfusion, or adoption of a mature blood or skeletal muscle cell phenotype.

As described and exemplified herein, the administration of PDNF or PDNFfragments can lead to an increased or decreased production of certaincytokines and paracrine factors that modulate immune response and reduceinflammation. It is generally believed that TH1 lymphocytes, although donot attack the myocardium themselves, could trigger cytotoxic T cells tobecome auto-reactive. These TH1-lymphocytes also produce high levels ofpro-inflammatory cytokines (such as IFN-γ, TNF-α, IL-1, IL-2, IL-12,IL-18, or IL-23), thereby suppressing the formation of regulatoryT-cells (Treg) and TH2. Inhibition of Treg formation allows a strongerauto-reactive cardiac response, thereby creating more cardiac damage.Contrary to TH1, TH2 do not enhance cytotoxic or phagocytic activity.Accordingly, their cytokine production is more immunosuppressive,including IL-10 and IL-4. Other immune-tolerant cytokines or factorsproduced by TH2 or Treg cells include, for example, TGF-β1, IL-5, IL-6,prostaglandin-E2 (PGE2).

It is believed that the endogenous stem cells described herein canmodulate T-cell response by altering the TH1/TH2 ratio. This leads to alarger percentage of Treg cells, which create an immuno-tolerantenvironment and more TH2 cells, suppressing the formation of TH1lymphocytes. Meanwhile, cell-mediated immune response via TH1 cells,recruiting more cytotoxic T cells and neutrophils, is also suppressed.

In another aspect, the invention provides a method of treating a subjectsuffering from, or susceptible to an inflammatory disease, such as acardiac or hepatic inflammatory disease, comprising: administering tosaid subject an amount of parasite-derived neurotrophic factor (PDNF),or a fragment of PDNF, where said fragment of PDNF binds to TrkAReceptor, and where the administration provides a serum concentrationof: at least about 1 ng/mL of IL-1ra, or at least about 0.5 ng/mL ofTSG-6 in said subject within 4 hours, 6 hours, 8 hours, 12 hours, 24hours, 48 hours, or 72 hours of administration. Another example of theinflammatory disease is an inflammatory disease of the GI-tract.

IL-1ra (interleukin-1 receptor antagonist) is a member of theinterleukin 1 cytokine family. IL1ra is secreted by various types ofcells including immune cells, epithelial cells, and adipocytes, and is anatural inhibitor of the pro-inflammatory effect of IL1β. This proteininhibits the activities of interleukin 1, alpha (IL1A) and interleukin1, beta (IL1B), and modulates a variety of interleukin 1 related immuneand inflammatory responses. In humans, IL1-ra is encoded by the IL1RNgene. It has been reported that the serum concentration of IL1-ra in anormal human is about 0.24-0.34 ng/ml) (see, e.g., Am. J. Respir. Crit.Care Med. 1997 April; 155(4):1469-73).

TSG-6 (Tumor necrosis factor-inducible gene 6 protein) is also known asTNF-stimulated gene 6 protein). In human, TSG-6 is encoded by theTNFAIP6 (tumor necrosis factor, alpha-induced protein 6) gene. Theexpression of this gene can be induced by a number of signalingmolecules, principally tumor necrosis factor α (TNF-α) and interleukin-1(IL-1). The expression can also be induced by mechanical stimuli invascular smooth muscle cells, and is found to be correlated withproteoglycan synthesis and aggregation. In addition, TSG-6 forms bothcovalent and non-covalent complexes with inter-α-inhibitor (a serineprotease inhibitor present at high levels in serum) and potentiates itsanti-plasmin activity. It has been reported that TSG-6 cannot bedetected in sera of normal individuals (see, e.g., Am J Pathol. 2001November; 159(5): 1711-1721).

In another aspect, the invention provides a method of treating a subjectsuffering from, or susceptible to an inflammatory disease, such as acardiac or hepatic inflammatory disease, comprising: administering tosaid subject an amount of parasite-derived neurotrophic factor (PDNF),or a fragment of PDNF, where said fragment of PDNF binds to TrkAReceptor, and where the administration provides a serum concentration ofat least about 0.25 ng/mL of Cox-2 in said subject within 4 hours, 6hours, 8 hours, 12 hours, 24 hours, 48 hours, or 72 hours ofadministration. Another example of the inflammatory disease is aninflammatory disease of the GI-tract.

COX-2 (cyclooxygenase-2 or prostaglandin-endoperoxide synthase 2) is anenzyme existing as a homodimer, each monomer with a molecular mass ofabout 70 kDa. In humans, COX-2 is encoded by the PTGS2 gene. COX-2 isunexpressed under normal conditions in most cells, but elevated levelsare found during inflammation. It has been reported that COX-2 isundetectable in most tissues in the absence of stimulation (see, e.g.,Proc. Natl. Acad. Sci. USA, Vol. 96, pp. 272-277, January 1999).

In another aspect, the invention provides a method of treating a subjectsuffering from, or susceptible to an inflammatory disease, comprising:administering to said subject an amount of parasite-derived neurotrophicfactor (PDNF), or a fragment of PDNF, where said fragment of PDNF bindsto TrkA Receptor, and where the administration provides a serumconcentration of: at least about 0.5 ng/mL, at least about 1 ng/mL, atleast about 1.5 ng/mL, at least about 2 ng/mL, at least about 2.5 ng/mL,at least about 3 ng/mL, at least about 3.5 ng/mL, at least about 4ng/mL, at least about 4.5 ng/mL, at least about 5 ng/mL, at least about5.5 ng/mL, or at least about 6 ng/mL, of IL1-ra within 4 hours, 6 hours,8 hours, 12 hours, 24 hours, 48 hours, or 72 hours of administration. Incertain embodiments, the inflammatory disease is mediated by theinterleukin-1 (IL-1) receptor. A non-exclusive list of acute and chronicinterleukin-1 (IL-1)-mediated inflammatory diseases includes but is notlimited to the following: acute pancreatitis; ALS; Alzheimer's disease;cachexia/anorexia; asthma; atherosclerosis; chronic fatigue syndrome,fever; diabetes (e.g., insulin diabetes); glomerulonephritis; graftversus host rejection; hemorrhagic shock; hyperalgesia, inflammatorybowel disease; inflammatory conditions of a joint, includingosteoarthritis, psoriatic arthritis and rheumatoid arthritis;degenerative disk disease; ischemic injury, including cerebral ischemia(e.g., brain injury as a result of trauma, epilepsy, hemorrhage orstroke, each of which may lead to neurodegeneration); lung diseases(e.g., ARDS); multiple myeloma; multiple sclerosis; myelogenous (e.g.,AML and CML) and other leukemias; myopathies (e.g., muscle proteinmetabolism, esp. in sepsis); osteoporosis; Parkinson's disease; pain;pre-term labor; psoriasis; reperfusion injury; septic shock; sideeffects from radiation therapy, temporal mandibular joint disease, tumormetastasis; or an inflammatory condition resulting from strain, sprain,cartilage damage, trauma, orthopedic surgery, infection or other diseaseprocesses.

In another aspect, the invention provides a method of treating a subjectsuffering from, or susceptible to an inflammatory disease, such as acardiac or hepatic inflammatory disease, comprising: administering tosaid subject an amount of parasite-derived neurotrophic factor (PDNF),or a fragment of PDNF, where said fragment of PDNF binds to TrkAReceptor, and where the administration provides: at least about 5 fold,at least about 6 fold, at least about 7 fold, at least about 8 fold, atleast about 9 fold, or at least about 10 fold increase of the serumconcentration of IL-1ra, as compared to serum concentration of IL-1raprior to administration, in said subject within 4 hours, 6 hours, 8hours, 12 hours, 24 hours, 48 hours, or 72 hours of administration.Another example of the inflammatory disease is an inflammatory diseaseof the GI-tract.

In another aspect, the invention provides a method of treating a subjectsuffering from, or susceptible to an inflammatory disease, comprising:administering to said subject an amount of parasite-derived neurotrophicfactor (PDNF), or a fragment of PDNF, where said fragment of PDNF bindsto TrkA Receptor, and where the administration provides a serumconcentration of: at least about 0.5 ng/mL, at least about 1 ng/mL, atleast about 1.5 ng/mL, at least about 2 ng/mL, at least about 2.5 ng/mL,at least about 3 ng/mL, at least about 3.5 ng/mL, at least about 4ng/mL, at least about 4.5 ng/mL, at least about 5 ng/mL, at least about5.5 ng/mL, or at least about 6 ng/mL, of TSG-6 within 4 hours, 6 hours,8 hours, 12 hours, 24 hours, 48 hours, or 72 hours of administration. Alink between TSG-6 and inflammation has been established by studies thathigh concentrations of TSG-6 are present in the synovial fluid ofpatients with rheumatoid arthritis and some other forms of arthritis,whereas no TSG-6 was detectable in control synovial fluids. Synovialcells from a rheumatoid arthritis patient showed high levels ofconstitutive TSG-6 production, and both synovial cells and chondrocytesin culture showed increased TSG-6 production in response to IL-1 orTNF-β (Wisniewski et al., supra, 1993). As such, the PDNF of PDNFfragments described herein can be used to treat inflammatory diseaseslinked to IL-1 or TNF-β, such as, rheumatoid arthritis, otherinflammatory connective tissue disorders, and cancer.

In another aspect, the invention provides a method of treating a subjectsuffering from, or susceptible to an inflammatory disease, comprising:administering to said subject an amount of parasite-derived neurotrophicfactor (PDNF), or a fragment of PDNF, where said fragment of PDNF bindsto TrkA Receptor, and where the administration provides a serumconcentration of: at least about 0.1 ng/mL, at least about 0.2 ng/mL, atleast about 0.25 ng/mL, at least about 0.3 ng/mL, at least about 0.4ng/mL, at least about 0.5 ng/mL, at least about 1 ng/mL, at least about1.5 ng/mL, at least about 2 ng/mL, at least about 2.5 ng/mL, at leastabout 3 ng/mL, at least about 3.5 ng/mL, at least about 4 ng/mL, atleast about 4.5 ng/mL, at least about 5 ng/mL, at least about 5.5 ng/mL,or at least about 6 ng/mL, of Cox-2 within 4 hours, 6 hours, 8 hours, 12hours, 24 hours, 48 hours, or 72 hours of administration.

The serum concentration of the inflammatory or anti-inflammatory markersdescribed herein, such as IL-1ra, TSG-6, and COX-2, can be determined byany method which quantitatively determines the amount of a molecule(s).One example is flow cytometry of a sample to quantitatively identify thepresence of the specific molecule in the sample. Another example is theuse an analytical system that utilizes multianalyte fluorometric beads,such as the Bead Lite Millipore luminex platform. Other assay methodsinclude, for example, quantitative immunology based assays such as ELISAanalysis, mass spectroscopy or mass spectrometry. The serumconcentrations of multiple cytokines or factors can be determinedsimultaneously, or separately.

The PDNF or a TrkA binding fragment thereof described herein may also beused to treat rheumatoid arthritis. Rheumatoid arthritis (RA) is anautoimmune disease characterized by chronic inflammation of synovialtissue, leading to destruction of the joint architecture. Efficacy ofthe administration can be measured by RA-associated biomarkers. Examplesof RA-associated biomarkers include, but are not limited to, e.g.,high-sensitivity C-reactive protein (hsCRP), serum amyloid A (SAA),erythrocyte sedimentation rate (ESR), serum hepcidin, interleukin-6(IL-6), and hemoglobin (Hb). As will be appreciated by a person ofordinary skill in the art, an increase or decrease in an RA-associatedbiomarker can be determined by comparing the level of the biomarkermeasured in the patient at a defined time point after administration ofPDNF (or fragments) to the level of the biomarker measured in thepatient prior to the administration (i.e., the “baseline measurement”).The defined time point at which the biomarker can be measured can be,e.g., at about 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days,35 days, 40 days or more after administration of PDNF (or PDNFfragments).

In another aspect, the invention provides a method of promoting theproliferation or mobilization of hepatic progenitor cells in a subjectin need thereof, comprising: administering to said subject atherapeutically effective amount of parasite-derived neurotrophic factor(PDNF), or a fragment of PDNF, where said PDNF fragment binds to TrkAReceptor.

In certain embodiments, the subject does not have Chagas disease. Incertain embodiments, the subject does not have a mycoplasma infection.In certain embodiments, the subject does not have a loss ofhematopoietic regeneration capacity.

Myocarditis and Cardiomyopathy

Myocarditis and cardiomyopathy are a group of diseases primarily of themyocardium that are not the result of hypertensive, congenital,ischemic, or valvular heart disease. Myocarditis generally refers to anacute myocardial disease characterized by inflammation, andcardiomyopathy generally refers to more chronic myocardial diseases inwhich the inflammatory features are not conspicuous (Concise Pathology,1st ed., Appleton & Lange, 367 (1991)). Cardiomyopathies can beclassified according to pathophysiologic type as dilated congestive,hypertrophic obstructive, hypertrophic non obstructive, apicalobliterative, diffuse nonobliterative restrictive, and obliterativerestrictive. Myocarditis and cardiomyopathy can lead to fever, chestpain, leukocytosis, increased erythrocyte sedimentation rate, leftventricular failure, arrhythmias, heart block, ECG changes, andeventually cardiac failure.

Myocarditis and cardiomyopathy result from an immune response againstthe myocardium, including lymphocytic infiltration and inflammation. Theimmune response can occur secondary to infectious diseases such asChagas disease (American trypanosomiasis), toxoplasmosis, trichinosis,rickettsial infection (typhus, Rocky Mountain spotted fever), fungalinfections, and metazoan parasites; or secondary to autoimmune diseasessuch as rheumatic fever, rheumatoid arthritis, systemic lupuserythematosus, progressive systemic sclerosis, and polyarthritis nodosa.The immune response leading to myocarditis can be idiopathic in natureas seen in Fiedler's myocarditis. Additionally, myocarditis can becaused by drug reaction to penicillin or sulfonamide, for example.

Acute endocarditis generally refers to an inflammatory disease of thevisceral or parietal pericardium (Pathology, J. B. Lippencott Co, 538(1988)), and can occur secondary to bacterial, viral (especiallyechovirus, and Coxsackie Group B), or fungal infection, and canaccompany systemic diseases such as rheumatoid arthritis, systemic lupuserythematosus, scleroderma, and uremia. Pericarditis can also occurafter cardiac trauma or cardiac surgery that is suggested as beingcaused by immunologic hypersensitivity. Acute pericarditis can lead tochronic constrictive pericarditis, effusion, and hemorrhage, all ofwhich can result in cardiac failure.

Myocardial Infarction (MI)

Myocardial infarction (MI) is the most common cause of mortality indeveloped countries. It is a multifactorial disease that involvesatherogenesis, thrombus formation and propagation. Thrombosis can resultin complete or partial occlusion of coronary arteries. The luminalnarrowing or blockage of coronary arteries reduces oxygen and nutrientsupply to the cardiac muscle (cardiac ischemia), leading to myocardialnecrosis and/or stunning. MI, unstable angina, and sudden ischemic deathare clinical manifestations of cardiac muscle damage. All threeendpoints are part of the Acute Coronary Syndrome. Recurrent myocardialinfarction can generally be viewed as a severe form of MI progressioncaused by multiple vulnerable plaques that are able to undergopre-rupture or a pre-erosive state, coupled with extreme bloodcoagulability.

Acute myocardial infarction is typically treated through a therapy inwhich blood flow is restored by recanalization of the occluded coronaryartery, together with treatment of arrhythmia, such as ventricularfibrillation. Myocardial ischemia-reperfusion therapies include athrombolytic therapy using, for example, t-PA, and PTCA using a ballooncatheter. However, it has been known that myocardialischemia-reperfusion causes inflammatory response due to, for example,free radicals (e.g., active oxygen), vascular endothelial cell injury,or neutrophil activation, resulting in additional damage to themyocardium. Thus, there is a need for development of a drug that reducesthe incidence of myocardial ischemia-reperfusion injury.

For example, after myocardial infarction, a local inflammatory reactionclears the damaged myocardium from dead cells and matrix debris at theonset of scar formation. The intensity and duration of this inflammatoryreaction are intimately linked to post-infarct remodeling and cardiacdysfunction. Strikingly, treatment with standard anti-inflammatory drugsworsens clinical outcome, suggesting a dual role of inflammation in thecardiac response to injury.

Stem Cell Therapies

Development of regenerative therapeutic strategies to reverse theprogression of advanced heart failure is one of the most urgent clinicalneeds. Cell replacement therapies using stem cells have attractedsignificant interest. For example, stem cell therapy using mesenchymalstem cells (MSCs) has been found to have beneficial effects, mostlyrelated to paracrine actions. F. van den Akker, et al., Cardiac stemcell therapy to modulate inflammation upon myocardial infarction,Biochimica et Biophysica Acta (2012),http://dx.doi.org/10.1016/j.bbagen.2012.08.026. One of the suggestedparacrine effects of stem cell therapy is modulation of the immunesystem. MSCs are reported to interact with several cells of the immunesystem and could therefore be a means to reduce detrimental inflammatoryreactions and promote the switch to the healing phase upon cardiacinjury.

Early studies in animals suggested that bone marrow cells might havepotential as cardiac regenerative therapeutics. Ptaszek, et al., Lancet379, 933-942 (2012). Many clinical trials followed these reports.Although injection with bone marrow cells was subsequently reported tobe safe, the associated benefit was variable. In some trials, benefitwas present, but short-lived. In other trials, no benefit was noted.Problems associated with the use of non-cardiac stem cells include, forexample, differentiation of the stem cells into non-cardiac cellsinstead of cardiomyocytes, inefficient delivery (˜10% cardiacretention), cell fusion, or adoption of a mature blood or skeletalmuscle cell phenotype.

For several decades, the paradigm of the heart being a terminallydifferentiated organ was accepted. Now there is a growing body ofevidence that challenges this dogma. It has been demonstrated that theheart contains a pool of stem cells that are not mobilized bone marrowcells, but actual stem cells residing in the heart (Beltrami et al.,Adult cardiac stem cells are multipotent and support myocardialregeneration, Cell, 2003; 114:763-76). Several reports describe thatcardiac stem cells can be isolated based on the expression of membraneantigens like c-kit, Sca-1 or Islet-1. See, e.g., Bearzi et al., Humancardiac stem cells, Proc Natl Acad Sci USA. 2007, 104:14068-73; Goumanset al., TGF-beta1 induces efficient differentiation of humancardiomyocyte progenitor cells into functional cardiomyocytes in vitro,Stem Cell Res. 2007, 1:138-49; Laugwitz et al., Postnatalisll+cardioblasts enter fully differentiated cardiomyocyte lineages,Nature. 2005, 433:647-53.

The identification of resident cardiac stem cells in the human heart,together with the isolation of a complex pool of cardiac cells (called“cardiospheres”) indicates a potential use of these autologous cells forcardiac repair, through the enhancement of endogenous regenerativeactivity. Increasing endogenous cardiac stem cells or cardiomyocytes canbe achieved with two approaches. One approach is to stimulate expansionof cardiomyocytes or cardiac stem cells with a drug or paracrine factor.The second approach involves propagation of cardiac cells withregenerative potential ex vivo followed by implantation of these cellsdirectly into an injured area. However, cell implantation afterinfarction can be limited by two factors: number of cells engrafted intothe recipient myocardium, and modest differentiation of thecardiomyocyte progeny. Further, the search for cardiac cells andparacrine factors that are capable of triggering cardiac repair has beenconsidered “challenging.” Ptaszek, et al., Lancet 379, 933-942 (2012).

The existence of resident cardiac stem cells provides a compellingstrategy for cardiac tissue repair. First, cardiac stem cells candifferentiate into cardiomyocytes, thereby promoting the regeneration ofthe damaged heart tissue. Second, cardiac stem cells can also reduce theinflammation of cardiac tissue. Often, when tissue damage occurs, aninflammatory response is stimulated to remove cell remnants and debris.For example, cardiac ischemic responses trigger a strong immune reactionin the heart. This reaction includes the activation of local macrophagesand the attraction of other immune cells from the blood, such asneutrophils, monocytes and lymphocytes. These immune cells produce alarge number of pro-inflammatory cytokines. This leads to a cascade inwhich more immune cells are attracted, causing further damage and stresson the surviving cardiomyocytes and leading to even more cell death.Cardiac stem cells can secrete paracrine factors, which can reduce celldeath, fibrosis and inflammation. Cardiac stem cells can also producegrowth factors and cytokines, and reduce or suppress the inflammatoryresponse that leads to tissue damage. As such, cardiac stem cells canreduce the detrimental inflammatory reactions upon cardiac injury, andpromote the switch to the healing phase. This anti-inflammatory effectof cardiac stem cells is surprising.

As disclosed and exemplified herein, the inventor discovered that PDNFis a paracrine factor that promotes both the proliferation andmobilization of stem cells. Although not wishing to be bound by aparticular theory, it is believed that PDNF increases the number ofendogenous resident cardiac stem cells by at least two mechanisms.First, it is believed that PDNF promotes the proliferation of cardiacstem cells (as shown by the increased expression of cardiac stem cellmarkers Sca-1 and c-Kit) by binding to TrkA receptor and activating theTrkA-mediated kinase pathway. Second, it is also believed that PDNF canpromote the mobilization of cardiac stem cells by increasing thesynthesis of chemotatic factors (e.g., an increased expression ofchemokine CCL2 and its receptor CCR2).

Accordingly, the invention is based in part on the surprising discoverythat PDNF can promote the proliferation and mobilization of cardiac stemcells. The increase in the resident cardiac stem cells, triggered byPDNF, in turn promotes the regeneration of damaged tissue, and reducesinflammatory response that can further damage the heart.

Use of the PDNF or PDNF fragment described herein for ex vivo therapy isalso envisioned. Because of the population of resident cardiac stemcells are very small, it might be important to expand the stem cellspopulation to take advantage of their regenerative and anti-inflammatoryactivities. Stem cells or progenitor cells can be isolated from thesubject or another donor, expanded in vitro in the presence of PDNF orPDNF fragment, and introduced to the subject. The stem cell populationcan be achieved by at least two means: promoting the proliferation ofthe stem cells (e.g., inducing the proliferation of quiescent stemcells), and enhancing the mobilization of stem cells (e.g., recruitingstem cells to the damaged heart tissue by chemotaxis).

It was also discovered that anti-inflammatory effect induced by PDNF isboth local (reduction of inflammation in heart) and systemic (reductionof inflammation in other tissues, such as liver). Therefore, thetherapeutic use of PDNF is not limited to cardiac inflammatory disease;instead, it can be used to treat inflammation in general.

Accordingly, as a stem cell renewal factor, PDNF can be used to treatinflammatory diseases, such as cardiac inflammatory diseases and hepaticinflammatory diseases. PDNF can also be used to treat an inflammatorydisease of the GI-tract.

Polynucleotide Therapy

Polynucleotide therapy is another therapeutic approach in which anucleic acid encoding a PDNF or sPDNF polypeptide is introduced intocells. The transgene is delivered to cells in a form in which it can betaken up and expressed in a therapeutically effective amount.

Transducing retroviral, adenoviral, or human immunodeficiency viral(HIV) vectors are used for somatic cell gene therapy because of theirhigh efficiency of infection and stable integration and expression (see,for example, Cayouette et al., Hum. Gene Ther., 8:423-430, 1997; Kido etal., Curr. Eye Res. 15:833-844, 1996; Bloomer et al., J. Virol.71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; Miyoshiet al., Proc. Natl. Acad. Sci. USA, 94:10319-10323, 1997). For example,PDNF or sPDNF nucleic acid molecules, or portions thereof, can be clonedinto a retroviral vector and driven from its endogenous promoter, fromthe retroviral long terminal repeat, or from a promoter specific for thetarget cell type of interest (such as epithelial carcinoma cells). Otherviral vectors that can be used include, but are not limited to,adenovirus, adeno-associated virus, vaccinia virus, bovine papillomavirus, vesicular stomatitus virus, or a herpes virus such asEpstein-Barr Virus.

Gene transfer can be achieved using non-viral means requiring infectionin vitro. This would include calcium phosphate, DEAE-dextran,electroporation, and protoplast fusion. Liposomes may also bepotentially beneficial for delivery of DNA into a cell. Although thesemethods are available, many of these are of lower efficiency.

Formulations

The administration of a compound or a combination of compounds for thetreatment of an inflammatory disorder may be by any suitable means thatresults in a concentration of the therapeutic that, combined with othercomponents, is effective in ameliorating, reducing, or stabilizing theinflammatory disorder. In one embodiment, a composition of the inventioncomprises a PDNF or sPDNF polypeptide. In another embodiment, acomposition of the invention comprises a cell contacted with PDNF orsPDNF polypeptide. The compound may be contained in any appropriateamount in any suitable carrier substance, and is generally present in anamount of 1-95% by weight of the total weight of the composition. Thecomposition may be provided in a dosage form that is suitable forparenteral (e.g., subcutaneously, intravenously, intramuscularly, orintraperitoneally) administration route. The pharmaceutical compositionsmay be formulated according to conventional pharmaceutical practice(see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.),ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopediaof Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York).

Human dosage amounts can initially be determined by extrapolating fromthe amount of compound used in mice, as a skilled artisan recognizes itis routine in the art to modify the dosage for humans compared to animalmodels. In certain embodiments it is envisioned that the dosage may varyfrom between about 1 μg compound/Kg body weight to about 5000 mgcompound/Kg body weight; or from about 5 mg/Kg body weight to about 4000mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kgbody weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg bodyweight; or from about 100 mg/Kg body weight to about 1000 mg/Kg bodyweight; or from about 150 mg/Kg body weight to about 500 mg/Kg bodyweight. In other embodiments this dose may be about 1, 5, 10, 25, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000,4500, or 5000 mg/Kg body weight. In other embodiments, it is envisagedthat doses may be in the range of about 5 mg compound/Kg body to about20 mg compound/Kg body. In other embodiments the doses may be about 8,10, 12, 14, 16 or 18 mg/Kg body weight. Of course, this dosage amountmay be adjusted upward or downward, as is routinely done in suchtreatment protocols, depending on the results of the initial clinicaltrials and the needs of a particular patient.

The precise determination of what would be considered an effective doseis based on factors individual to each subject, including their size,age, sex, weight, and condition of the particular subject. Dosages canbe readily ascertained by those skilled in the art from this disclosureand the knowledge in the art.

Optionally, the methods of the invention provide for the administrationof a composition of the invention to a suitable animal model to identifythe dosage of the composition(s), concentration of components thereinand timing of administering the composition(s), which elicit tissuerepair, reduce cell death, or induce another desirable biologicalresponse. Such determinations do not require undue experimentation, butare routine and can be ascertained without undue experimentation.

The biologically active agents can be conveniently provided to a subjectas sterile liquid preparations, e.g., isotonic aqueous solutions,suspensions, emulsions, dispersions, or viscous compositions, which maybe buffered to a selected pH. Cells and agents of the invention may beprovided as liquid or viscous formulations. For some applications,liquid formations are desirable because they are convenient toadminister, especially by injection. Where prolonged contact with atissue is desired, a viscous composition may be preferred. Suchcompositions are formulated within the appropriate viscosity range.Liquid or viscous compositions can comprise carriers, which can be asolvent or dispersing medium containing, for example, water, saline,phosphate buffered saline, polyol (for example, glycerol, propyleneglycol, liquid polyethylene glycol, and the like) and suitable mixturesthereof.

Sterile injectable solutions are prepared by suspending PDNF or sPDNFpolypeptide in the required amount of the appropriate solvent withvarious amounts of the other ingredients, as desired. Such compositionsmay be in admixture with a suitable carrier, diluent, or excipient, suchas sterile water, physiological saline, glucose, dextrose, or the like.The compositions can also be lyophilized. The compositions can containauxiliary substances such as wetting, dispersing, or emulsifying agents(e.g., methylcellulose), pH buffering agents, gelling or viscosityenhancing additives, preservatives, flavoring agents, colors, and thelike, depending upon the route of administration and the preparationdesired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”,17th edition, 1985, incorporated herein by reference, may be consultedto prepare suitable preparations, without undue experimentation.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin. According to the present invention,however, any vehicle, diluent, or additive used would have to becompatible with the cells or agents present in their conditioned media.

The compositions can be isotonic, i.e., they can have the same osmoticpressure as blood and lacrimal fluid. The desired isotonicity of thecompositions of this invention may be accomplished using sodiumchloride, or other pharmaceutically acceptable agents such as dextrose,boric acid, sodium tartrate, propylene glycol or other inorganic ororganic solutes. Sodium chloride is preferred particularly for bufferscontaining sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent,such as methylcellulose. Other suitable thickening agents include, forexample, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose,carbomer, and the like. The choice of suitable carriers and otheradditives will depend on the exact route of administration and thenature of the particular dosage form, e.g., liquid dosage form (e.g.,whether the composition is to be formulated into a solution, asuspension, gel or another liquid form, such as a time release form orliquid-filled form). Those skilled in the art will recognize that thecomponents of the compositions should be selected to be chemicallyinert.

Methods of Delivery

Compositions comprising PDNF or sPDNF may be delivered to a subject inneed thereof. Modes of administration include intramuscular,intra-cardiac, intra-hepatic, oral, rectal, topical, intraocular,buccal, intravaginal, intracisternal, intra-arterial,intracerebroventricular, intratracheal, nasal, transdermal, within/onimplants, e.g., fibers such as collagen, osmotic pumps, or parenteralroutes. The term “parenteral” includes subcutaneous, intravenous,intramuscular, intraperitoneal, intragonadal or infusion.

The compositions can be administered via localized injection, includingcatheter administration, systemic injection, localized injection,intravenous injection, or parenteral administration. When administeringa therapeutic composition of the present invention, it will generally beformulated in a unit dosage injectable form (solution, suspension,emulsion). Dosages can be readily adjusted by those skilled in the art(e.g., a decrease in purity may require an increase in dosage).Compositions of the invention can be introduced by injection, catheter,or the like. Compositions of the invention include pharmaceuticalcompositions comprising cellular factors of the invention and apharmaceutically acceptable carrier. Administration can be autologous orheterologous.

Pharmaceutical compositions according to the invention may be formulatedto release the active compound substantially immediately uponadministration or at any predetermined time or time period afteradministration. The latter types of compositions are generally known ascontrolled release formulations, which include (i) formulations thatcreate a substantially constant concentration of the drug within thebody over an extended period of time; (ii) formulations that after apredetermined lag time create a substantially constant concentration ofthe drug within the body over an extended period of time; (iii)formulations that sustain action during a predetermined time period bymaintaining a relatively, constant, effective level in the body withconcomitant minimization of undesirable side effects associated withfluctuations in the plasma level of the active substance (sawtoothkinetic pattern); (iv) formulations that localize action by, e.g.,spatial placement of a controlled release composition adjacent to or incontact with the thymus; (v) formulations that allow for convenientdosing, such that doses are administered, for example, once every one ortwo weeks; and (vi) formulations that target a neoplasia by usingcarriers or chemical derivatives to deliver the therapeutic agent to aparticular cell type (e.g., islet or beta cell). For some applications,controlled release formulations obviate the need for frequent dosingduring the day to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the compound in question. In one example, controlledrelease is obtained by appropriate selection of various formulationparameters and ingredients, including, e.g., various types of controlledrelease compositions and coatings. Thus, the therapeutic is formulatedwith appropriate excipients into a pharmaceutical composition that, uponadministration, releases the therapeutic in a controlled manner.Examples include single or multiple unit tablet or capsule compositions,oil solutions, suspensions, emulsions, microcapsules, microspheres,molecular complexes, nanoparticles, patches, and liposomes.

Parenteral Compositions

The pharmaceutical composition may be administered parenterally byinjection, infusion or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non-toxic pharmaceutically acceptable carriers andadjuvants. The formulation and preparation of such compositions are wellknown to those skilled in the art of pharmaceutical formulation.Formulations can be found in Remington: The Science and Practice ofPharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in the form of a solution, a suspension, an emulsion,an infusion device, or a delivery device for implantation, or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active agent that reduces orameliorates a neoplasia, the composition may include suitableparenterally acceptable carriers and/or excipients. The activetherapeutic agent(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. Furthermore, the composition may include suspending,solubilizing, stabilizing, pH-adjusting agents, tonicity adjustingagents, and/or dispersing, agents.

As indicated above, the pharmaceutical compositions according to theinvention may be in the form suitable for sterile injection. To preparesuch a composition, the suitable active therapeutic(s) are dissolved orsuspended in a parenterally acceptable liquid vehicle. Among acceptablevehicles and solvents that may be employed are water, water adjusted toa suitable pH by addition of an appropriate amount of hydrochloric acid,sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer'ssolution, and isotonic sodium chloride solution and dextrose solution.The aqueous formulation may also contain one or more preservatives(e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where oneof the compounds is only sparingly or slightly soluble in water, adissolution enhancing or solubilizing agent can be added, or the solventmay include 10-60% w/w of propylene glycol or the like.

Controlled Release Parenteral Compositions

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. Alternatively, the active drugmay be incorporated in biocompatible carriers, liposomes, nanoparticles,implants, or infusion devices.

Materials for use in the preparation of microspheres and/ormicrocapsules are, e.g., biodegradable/bioerodible polymers such aspolygalactia poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutam-nine) and, poly(lactic acid).

Biocompatible carriers that may be used when formulating a controlledrelease parenteral formulation are carbohydrates (e.g., dextrans),proteins (e.g., albumin), lipoproteins, or antibodies. Materials for usein implants can be non-biodegradable (e.g., polydimethyl siloxane) orbiodegradable (e.g., poly(caprolactone), poly(lactic acid),poly(glycolic acid) or poly(ortho esters) or combinations thereof).

Solid Dosage Forms for Oral Use

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. Such formulations are known to the skilled artisan.Excipients may be, for example, inert diluents or fillers (e.g.,sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starchesincluding potato starch, calcium carbonate, sodium chloride, lactose,calcium phosphate, calcium sulfate, or sodium phosphate); granulatingand disintegrating agents (e.g., cellulose derivatives includingmicrocrystalline cellulose, starches including potato starch,croscarmellose sodium, alginates, or alginic acid); binding agents(e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodiumalginate, gelatin, starch, pregelatinized starch, microcrystallinecellulose, magnesium aluminum silicate, carboxymethylcellulose sodium,methylcellulose, hydroxypropyl methylcellulose, ethylcellulose,polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents,glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate,stearic acid, silicas, hydrogenated vegetable oils, or talc). Otherpharmaceutically acceptable excipients can be colorants, flavoringagents, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active drug ina predetermined pattern (e.g., in order to achieve a controlled releaseformulation) or it may be adapted not to release the active drug untilafter passage of the stomach (enteric coating). The coating may be asugar coating, a film coating (e.g., based on hydroxypropylmethylcellulose, methylcellulose, methyl hydroxyethyl cellulose,hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers,polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating(e.g., based on methacrylic acid copolymer, cellulose acetate phthalate,hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcelluloseacetate succinate, polyvinyl acetate phthalate, shellac, and/orethylcellulose). Furthermore, a time delay material, such as, e.g.,glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active anti-neoplasiatherapeutic substance). The coating may be applied on the solid dosageform in a similar manner as that described in Encyclopedia ofPharmaceutical Technology, supra.

At least two anti-neoplasia therapeutics may be mixed together in thetablet, or may be partitioned. In one example, the first activeanti-neoplasia therapeutic is contained on the inside of the tablet, andthe second active anti-neoplasia therapeutic is on the outside, suchthat a substantial portion of the second anti-neoplasia therapeutic isreleased prior to the release of the first anti-neoplasia therapeutic.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, lactose, microcrystallinecellulose, calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example, peanut oil, liquid paraffin, or olive oil.Powders and granulates may be prepared using the ingredients mentionedabove under tablets and capsules in a conventional manner using, e.g., amixer, a fluid bed apparatus or a spray drying equipment.

Controlled Release Oral Dosage Forms

Controlled release compositions for oral use may, e.g., be constructedto release the active anti-neoplasia therapeutic by controlling thedissolution and/or the diffusion of the active substance. Dissolution ordiffusion controlled release can be achieved by appropriate coating of atablet, capsule, pellet, or granulate formulation of compounds, or byincorporating the compound into an appropriate matrix. A controlledrelease coating may include one or more of the coating substancesmentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax,carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryldistearate, glycerol palmitostearate, ethylcellulose, acrylic resins,dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride,polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate,methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydro gels, 1,3butylene glycol, ethylene glycol methacrylate, and/or polyethyleneglycols. In a controlled release matrix formulation, the matrix materialmay also include, e.g., hydrated methylcellulose, carnauba wax andstearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

A controlled release composition containing one or more therapeuticcompounds may also be in the form of a buoyant tablet or capsule (i.e.,a tablet or capsule that, upon oral administration, floats on top of thegastric content for a certain period of time). A buoyant tabletformulation of the compound(s) can be prepared by granulating a mixtureof the compound(s) with excipients and 20-75% w/w of hydrocolloids, suchas hydroxyethylcellulose, hydroxypropylcellulose, orhydroxypropylmethylcellulose. The obtained granules can then becompressed into tablets. On contact with the gastric juice, the tabletforms a substantially water-impermeable gel barrier around its surface.This gel barrier takes part in maintaining a density of less than one,thereby allowing the tablet to remain buoyant in the gastric juice.

Combination Therapy

The PDNF or PDNF fragments described herein may be used together withone or more additional therapeutic agents. When administered incombination, each component may be administered at the same time, orsequentially in any order at different points in time.

Exemplary therapeutic agents include anti-coagulant or coagulationinhibitory agents, anti-platelet or platelet inhibitory agents, thrombininhibitors, Vitamin K antagonists, glycoprotein IIb/IIIa receptorantagonists, thrombolytic or fibrinolytic agents, anti-arrhythmicagents, anti-hypertensive agents, angiotensin converting enzymeinhibitors (ACE-Is), angiotensin receptor blockers (ARBs),beta-blockers, calcium channel blockers (L-type and T-type), cardiacglycosides, diruetics, mineralocorticoid receptor antagonists,phospodiesterase inhibitors, cholesterol/lipid lowering agents and lipidprofile therapies, anti-diabetic agents, anti-depressants,anti-inflammatory agents (steroidal and non-steroidal),anti-osteoporosis agents, hormone replacement therapies, oralcontraceptives, anti-obesity agents, anti-anxiety agents,anti-proliferative agents, anti-tumor agents, anti-ulcer andgastroesophageal reflux disease agents, growth hormone and/or growthhormone secretagogues, thyroid mimetics (including thyroid receptorantagonist), anti-infective agents, anti-viral agents, anti-bacterialagents, and anti-fungal agents.

Commonly used anti-inflammatory agents include, for example,non-steroidal anti-inflammatory drugs (NSAIDS) such as ibuprofen,naproxen, sulindac, indomethacin, mefenamate, droxicam, diclofenac,sulfinpyrazone, piroxicam, and pharmaceutically acceptable salts orprodrugs thereof. Of the NSAIDS, aspirin (acetylsalicyclic acid or ASA)and piroxicam are more commonly used. Anti-inflammatory agents include,without limitation, Alclofenac; Alclometasone Dipropionate; AlgestoneAcetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium;Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone;Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride;Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone;Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac;Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort;Desonide; Desoximetasone; Dexamethasone Dipropionate; DiclofenacPotassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac;Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap;Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac;Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; and Zomepirac Sodium.

TNF is a molecule involved in the inflammatory response of patients withinflammatory diseases. Accordingly, any molecule that blocks TNFfunction e.g., by blocking TNF binding to the TNF receptor (TNFR), mayhelp modify the progression of an inflammatory disease and alleviatesome of its symptoms. Several TNF blockers such as infliximab andetanercept, have been shown to be efficacious in treating cardiovasculardiseases.

Molecule that blocks the function of a pro-inflammatory cytokine, e.g.,by blocking IL-1 interaction with its receptor, may help modify theprogression of inflammatory diseases and alleviate one or more symptoms.Anakinra, a recombinant protein that blocks IL-1 interaction with itsreceptor (IL-1R) has been shown to be efficacious in treatingcardiovascularoid arthritis.

Methods for Evaluating Therapeutic Efficacy

In one approach, the efficacy of the treatment is evaluated bymeasuring, for example, the biological function of the treated organ(e.g., cardiac cell function). Such methods are standard in the art andare described, for example, in the Textbook of Medical Physiology, Tenthedition, (Guyton et al., W.B. Saunders Co., 2000). In particular, amethod of the present invention, increases the biological function of atissue or organ by at least 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 150%, 200%, or even by as much as 300%, 400%, or 500%. Preferably,the tissue is cardiac tissue and, preferably, the organ is heart.

In another approach, the therapeutic efficacy of the methods of theinvention is assayed by measuring an increase in cell number in thetreated or transplanted tissue or organ as compared to a correspondingcontrol tissue or organ (e.g., a tissue or organ that did not receivetreatment). Preferably, cell number in a tissue or organ is increased byat least 5%, 10%, 20%, 40%, 60%, 80%, 100%, 150%, or 200% relative to acorresponding tissue or organ. Methods for assaying cell proliferationare known to the skilled artisan and are described, for example, inBonifacino et al., (Current Protocols in Cell Biology Loose-leaf, JohnWiley and Sons, Inc., San Francisco, Calif.). For example, assays forcell proliferation may involve the measurement of DNA synthesis duringcell replication. In one embodiment, DNA synthesis is detected usinglabeled DNA precursors, such as [³H]-Thymidine or5-bromo-2*-deoxyuridine [BrdU], which are added to cells (or animals)and then the incorporation of these precursors into genomic DNA duringthe S phase of the cell cycle (replication) is detected (Ruefli-Brasseet al., Science 302(5650):1581-4, 2003; Gu et al., Science 302(5644):445-9, 2003).

In another approach, efficacy is measured by detecting an increase inthe number of viable cells present in a tissue or organ relative to thenumber present in an untreated control tissue or organ, or the numberpresent prior to treatment. Assays for measuring cell viability areknown in the art, and are described, for example, by Crouch et al. (J.Immunol. Meth. 160, 81-8); Kangas et al. (Med. Biol. 62, 338-43, 1984);Lundin et al., (Meth. Enzymol. 133, 27-42, 1986); Petty et al.(Comparison of J. Biolum. Chemilum. 10, 29-34, 0.1995); and Cree et al.(AntiCancer Drugs 6: 398-404, 1995). Cell viability can be assayed usinga variety of methods, including MTT(3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) (Barltrop,Bioorg. & Med. Chem. Lett. 1: 611, 1991; Cory et al., Cancer Comm. 3,207-12, 1991; Paull J. Heterocyclic Chem. 25, 911, 1988). Assays forcell viability are also available commercially. These assays include butare not limited to CELLTITER-GLO® Luminescent Cell Viability Assay(Promega), which uses luciferase technology to detect ATP and quantifythe health or number of cells in culture, and the CellTiter-Glo®Luminescent Cell Viability Assay, which is a lactate dehydrogenase (LDH)cytotoxicity assay (Promega).

Alternatively, or in addition, therapeutic efficacy is assessed bymeasuring a reduction in apoptosis. Apoptotic cells are characterized bycharacteristic morphological changes, including chromatin condensation,cell shrinkage and membrane blebbing, which can be clearly observedusing light microscopy. The biochemical features of apoptosis includeDNA fragmentation, protein cleavage at specific locations, increasedmitochondrial membrane permeability, and the appearance ofphosphatidylserine on the cell membrane surface. Assays for apoptosisare known in the art. Exemplary assays include TUNEL (Terminaldeoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) assays,caspase activity (specifically caspase-3) assays, and assays forfas-ligand and annexin V. Commercially available products for detectingapoptosis include, for example, Apo-ONE® Homogeneous Caspase-3/7 Assay,FragEL TUNEL kit (ONCOGENE RESEARCH PRODUCTS, San Diego, Calif.), theApoBrdU DNA Fragmentation Assay (BIOVISION, Mountain View, Calif.), andthe Quick Apoptotic DNA Ladder Detection Kit (BIOVISION, Mountain View,Calif.).

Methods for Evaluating Cardiac Function

Compositions of the invention may be used to enhance cardiac function ina subject having reduced cardiac function. Methods for measuring thebiological function of the heart (e.g., contractile function) arestandard in the art and are described, for example, in the Textbook ofMedical Physiology, Tenth edition, (Guyton et al., W.B. Saunders Co.,2000). In the invention, cardiac function is increased by at least 5%,10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% relativeto the cardiac function present in a naturally-occurring, correspondingtissue or organ. Most advantageously, cardiac function is enhanced ordamage is reversed, such that the function is substantially normal(e.g., 85%, 90%, 95%, or 100% of the cardiac function of a healthycontrol subject). Reduced cardiac function may result from conditionssuch as cardiac hypertrophy, reduced systolic function, reduceddiastolic function, maladaptive hypertrophy, heart failure withpreserved systolic function, diastolic heart failure, hypertensive heartdisease, aortic and mitral valve disease, pulmonary valve disease,hypertrophic cardiomyopathy (e.g., hypertrophic cardiomyopathyoriginating from a genetic or a secondary cause), post ischemic andpost-infarction cardiac remodeling and cardiac failure.

Any number of standard methods are available for assaying cardiovascularfunction. Preferably, cardiovascular function in a subject (e.g., ahuman) is assessed using non-invasive means, such as measuring netcardiac ejection (ejection fraction, fractional shortening, andventricular end-systolic volume) by an imaging method suchechocardiography, nuclear or radiocontrast ventriculography, or magneticresonance imaging, and systolic tissue velocity as measured by tissueDoppler imaging. Systolic contractility can also be measurednon-invasively using blood pressure measurements combined withassessment of heart outflow (to assess power), or with volumes (toassess peak muscle stiffening). Measures of cardiovascular diastolicfunction include ventricular compliance, which is typically measured bythe simultaneous measurement of pressure and volume, early diastolicleft ventricular filling rate and relaxation rate (can be assessed fromechoDoppler measurements). Other measures of cardiac function includemyocardial contractility, resting stroke volume, resting heart rate,resting cardiac index (cardiac output per unit of time [L/minute],measured while seated and divided by body surface area [m²])) totalaerobic capacity, cardiovascular performance during exercise, peakexercise capacity, peak oxygen (O₂) consumption, or by any other methodknown in the art or described herein. Measures of vascular functioninclude determination of total ventricular afterload, which depends on anumber of factors, including peripheral vascular resistance, aorticimpedance, arterial compliance, wave reflections, and aortic pulse wavevelocity, Methods for assaying cardiovascular function include any oneor more of the following: Doppler echocardiography, 2-dimensionalecho-Doppler imaging, pulse-wave Doppler, continuous wave Doppler,oscillometric arm cuff, tissue Doppler imaging, cardiac catheterization,magnetic resonance imaging, positron emission tomography, chest X-ray, Xray contrast ventriculography, nuclear imaging ventriculography,computed tomography imaging, rapid spiral computerized tomographicimaging, 3-D echocardiography, invasive cardiac pressures, invasivecardiac flows, invasive cardiac cardiac pressure-volume loops(conductance catheter), non-invasive cardiac pressure-volume loops.

Kits

The present compositions may be assembled into kits or pharmaceuticalsystems for use in ameliorating an inflammatory disease or disorder.Compositions of the invention comprising biologically active agents(e.g., PDNF or sPDNF) are supplied along with additional reagents in akit. The kits can include instructions for the treatment regime,reagents, equipment (test tubes, reaction vessels, needles, syringes,etc.) and standards for calibrating or conducting the treatment. Theinstructions provided in a kit according to the invention may bedirected to suitable operational parameters in the form of a label or aseparate insert. Optionally, the kit may further comprise a standard orcontrol information so that the test sample can be compared with thecontrol information standard to determine if whether a consistent resultis achieved.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, or other container means, into which acomponent may be placed, and preferably, suitably aliquoted. Where thereis more than one component in the kit, the kit also will generallycontain additional containers into which the additional components maybe separately placed. However, various combinations of components may becomprised in a container. The kits of the present invention also willtypically include a means for packaging the component containers inclose confinement for commercial sale. Such packaging may includeinjection or blow-molded plastic containers into which the desiredcomponent containers are retained.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1. Parasite-Derived Neurotrophic Factor (PDNF) PromotesStem Cell Activity and Proliferation

This example shows that PDNF is a renewal factor for cardiac stem cellsand hepatic stem cell. In addition, PDNF stimulated the expression ofanti-inflammatory factors IL1-Ra, TSG-6 and COX-2.

As shown in FIGS. 2A-2F, intravenous administration of soluble PDNF(sPDNF) into naïve mice increased expression of cardiac and hepatic stemcell markers independently of TLR signaling, expanded cardiac Sca-1⁺cells, and increased expression of IL1-Ra, TSG-6 and COX-2.

FIGS. 3A and 3B show that intravenous administration of soluble PDNF(sPDNF) triggered a dramatic reduction in inflammatory infiltrates inthe heart of mice with chronic Chagas cardiomyopathy (CCC).

FIGS. 4A-4C show that intravenous administration of soluble PDNF (sPDNF)reduced cardiac fibrosis in mice with chronic Chagas cardiomyopathy(CCC).

FIGS. 5A-5F show that intravenous administration of soluble PDNF (sPDNF)triggered a global decrease in the transcripts of cardiac inflammatoryand fibrogenic markers, and increased expression of theanti-inflammatory TSG-6 in the heart and liver of mice with chronicChagas cardiomyopathy (CCC).

Example 2. Use of sPDNF for Treatment of Inflammatory Bowel Disease(IBD)

In this example, a mouse model of IDB is used, and the regenerativechemotherapeutic profile is determined to assess the effect of sPDNFadministration.

Rag 2^(−/−) mice (from Jackson Laboratories) are administeredintraperitoneally 1×10⁵ CD4⁺CD25⁻ splenic T cells from C57BL/6 mice(from Jackson Laboratories). CD4⁺CD25⁻ T cells are isolated byfluorescence cell sorting. One week after T cell transfer, Rag mice areadministered piroxicam, a nonsteroidal anti-inflammatory drug (NSAID),mixed into their feed for 2 weeks (42 mg piroxicam/250 g chow, week 1;62 mg piroxicam/250 g chow, week 2). The piroxicam (Sigma-Aldrich) isstopped, and colitis is studied 1 week later. Intraperitoneal (i.p.)injection of sPDNF is given in accord with the scheme as shown in FIG.6. Animals are sacrificed 4 weeks from the day of cell transfer. Colonsare isolated, and half of the colons divided longitudinally are fixed,sectioned, and stained with H&E for microscopic examination to score theseverity of colitis. The other half is dissociated with collagenase toisolate lamina propria mononuclear cells (LPMC), which are analyzed byflow cytometry and cultured in vitro to determine levels of inflammatorycytokines.

Example 3. T. cruzi and sPDNF Augmented Expression of MCP-1 and FKN inCardiomyocytes and Cardiac Fibroblasts

Whether T. cruzi and sPDNF augment the expression of chemokines incardiomyocytes and cardiac fibroblasts was determined, focusing on MCP-1and FKN. In line with, and extending an earlier study (26), T. cruziinfection of primary cardiomyocytes and cardiac fibroblasts increasedexpression of MCP-1 and FKN transcripts in a time-dependent manner(FIGS. 7A and 7B). In addition, the stimulatory activity of T. cruzi wasmimicked by sPDNF, which stimulated an increase in MCP-1 and FKNtranscripts in both primary cardiomyocytes and cardiac fibroblasts in adose-dependent manner (FIGS. 7B and 7C).

In the cardiomyocyte cell line H9c2, sPDNF dramatically increasedsecretion of MCP-1 dose-dependently (FIG. 8A) and specifically, as sPDNFdid not alter secretion of another chemokine, macrophage inflammatoryprotein-2a/MIP-2a/CXCL2, and of the cytokine IL-6 (FIG. 8A). Similar tothe results obtained with primary cardiomyocytes, the increase in MCP-1and FKN mRNA in H9c2 cardiomyocytes was dose-dependent (FIG. 8B).Furthermore, time-course analysis revealed that MCP-1 transcriptincreased maximally 3 hr after sPDNF stimulation and dropped sharply tolevels similar to unstimulated cells after 8 hr, with typical pulse-likekinetics (FIG. 8C).

Example 4. Targeting Cardiomyocyte Neurotrophin Receptors TrkA and TrkCIncreased Expression of MCP-1 and FKN

Three distinct characteristics were used to determine whether T. cruziPDNF exploits TrkA or TrkC, or both, to increase MCP-1 and FKNproduction in cardiac cells. First, H9c2 cardiomyocytes werepre-incubated (1 hr) with the Trk antagonist K252a, which blocks Trksignaling by inhibiting Trk autophosphorylation (53), followed bystimulation (3 hr) with sPDNF (1 μg/ml) or vehicle PBS, andquantification of MCP-1 and FKN transcripts by qPCR. K252a completelyabrogated the stimulatory effect of sPDNF (FIG. 9A and FIG. 9B),indicating that the agonistic effect of sPDNF required Trk signaling.

Second, cardiomyocytes were pre-incubated (0.5 hr) with neutralizingantibodies against neurotrophin receptors prior to stimulation withsPDNF (1 μg/ml, 3 hr). Without being bound to a particular theory, ifsPDNF uses Trk receptors to augment chemokine expression, thenneutralizing the receptors with antibodies against the extracellulardomain of the receptors will prevent access of sPDNF to the receptorsand block sPDNF stimulation of MCP-1 and FKN expression. Antibodiesagainst TrkA (α-TrkA+sPDNF) significantly blocked sPDNF-inducedupregulation (+sPDNF) of MCP-1 (29.2±3.5% inhibition) and FKN (29.5±5.3%inhibition), as did antibodies against TrkC (MCP-1 inhibition, 53±2.3%;FKN inhibition, 47.5±1.3%) (FIGS. 9C and 9D). In contrast, neutralizingantibodies against TrkB (α-TrkB+sPDNF) were ineffective in preventingsPDNF upregulation of both MCP-1 and FKN (FIGS. 9C and 9D), consistentwith the previous finding that TrkB was not recognized by sPDNF, thusserving as a negative control for the α-TrkA and α-TrkC inhibitionexperiments (12, 59).

Third, experiments were designed to reduce Trk expression with shorthairpin mRNA (shRNA) to further validate the conclusion that enhancedexpression of MCP-1 and FKN resulted from the binding of sPDNF to TrkAand TrkC. For this purpose H9c2 cardiomyocytes were transfected withlentivirus encoding shRNA for control green fluorescence protein(shGFP), TrkA (four distinct vectors), or TrkC (five distinct vectors).Untransfected and transfected myocytes were stimulated with sPDNF (1μg/ml, 3 hr), and the inhibitory effect of gene silencing was assessedby comparing secreted MCP-1 levels in sPDNF-stimulated cells with thoseof vehicle PBS-treated cells. Compared to GFP-transfected cardiomyocytesor untransfected cardiomyocytes, cardiomyocytes transfected with shTrkAor shTrkC constructs significantly blocked MCP-1 secretion in responseto sPDNF stimulation (FIG. 9E).

Thus, on the basis of these three characteristics that operate bydistinct mechanisms, it was concluded that sPDNF used both TrkA and TrkCto increase expression of MCP-1 and FKN, akin to the utilization of TrkAby the matricellular protein CTGF/CCN2 to increase expression MCP-1 incardiomyocytes (58). Notably, dual usage of TrkA and TrkC to increaseexpression of MCP-1 and FKN is analogous to neuronal cell invasion,which depends on both TrkA (18) and TrkC (60). However, dual usage ofTrkA and TrkC to increase expression of MCP-1 and FKN was contrary tothe mechanism of T. cruzi entry into cardiac fibroblasts orcardiomyocytes, which depends on TrkC (5), and contrary to sPDNF-inducedNGF secretion by cardiomyocytes, which is selective for TrkA (4).

Example 5. Cardiac MCP-1 and FKN have Increased Expression in a MouseModel of Acute Chagas Myocarditis

Groups of three mice were infected with T. cruzi (Tulahuen strain),sacrificed at several days post-infection (PI). Cardiac MCP-1 and FKNmRNAs and cardiac parasite burden were quantified by qPCR (4, 5, 17, 18,60). In line with a previous study (34), the levels of MCP-1 and FKNmRNA paralleled the degree of heart parasite burden (FIGS. 10A and 10B).However, unlike MCP-1, expression of FKN transcripts remainedsignificantly elevated for at least 20 days after cardiac parasitismbecomes barely detectable (FIG. 10B). Without being bound to aparticular theory, this result is in agreement with the view that FKNplays an important role in recruiting blood monocytes that promotecardiac healing after myocardial injury (33).

Example 6. A Single Dose of Intravenous sPDNF Increased MCP-1 and FKNExpression in the Heart Dose-Dependently and with Pulse-Like Kinetics inBoth Naive Wild Type and MyD88^(−/−) Mice

The robust MCP-1 increase in acutely infected hearts (FIG. 10A) waslikely due to the stimulation of TLRs and other canonicalpro-inflammatory pathways by T. cruzi molecules, exemplified byglycosylphosphatidylinositol-anchored mucin-like glycoproteins.GPI-anchored mucin-like glycoproteins increase MCP-1 and mediateleukocyte recruitment in the pleural cavity of mice primed with anIFN-γ-inducing agent (16). Without being bound to a particular theory,if T. cruzi-PDNF is also a mechanism responsible for triggering MCP-1expression in T cruzi-infected hearts, then intravenous (IV)administration of sPDNF should increase expression of chemokines MCP-1and FKN in the heart of uninfected, naive mice. (FIGS. 10A and 10B). Aprevious pharmacokinetics study showed that sPDNF administered IV intonaive mice has a half-life of −15 min in the blood and peaks in themyocardium ˜15 min post-injection, triggering cardiac responses such asNGF upregulation hours after injection (4).

Groups of naive wild type C57BL/6 mice were injected with vehicle (PBS)or a single dose of IV sPDNF. Mice were sacrificed at various timepointspost-injection, and heart MCP-1 and FKN expression measured by real timePCR. Administration of IV sPDNF significantly increased expression ofboth cardiac MCP-1 and FKN transcripts with pulse-like kinetics. Bothchemokines peaked shortly after administration (3 hr post-injection) andquickly decreased to levels observed in un-injected mice (FIG. 11A). Theagonist effect of IV sPDNF was screened in various organs using optimumtime response (3 hr post-injection) using the same dose as in FIG. 11A.Treatment with sPDNF increased expression of MCP-1 not only in theheart, liver (where the response was most robust) and spleen, but not inthe bone marrow, aorta, esophagus, colon and skin (FIG. 11B). Organdistribution of MCP-1 expression in response to IV sPDNF was similar tothat of FKN. Without being bound to a particular theory, the selectiveincrease of MCP-1 and FKN in various organs may reflect the unevenexpression of PDNF receptors TrkA and TrkC in neural and non-neuraltissues (22, 50). Furthermore, increased expression of MCP-1 and FKNtriggered by IV sPDNF was dose-dependent in the heart (FIG. 11C) andliver (FIG. 11D). Without being bound to a particular theory, thisresult is in agreement with the hypothesis that sPDNF stimulates MCP-1and FKN through a receptor (TrkA and TrkC)-mediated process, akin to theresults obtained for cardiac cells in culture (FIGS. 9A-9E).

The sPDNF preparations did not have detectable levels of endotoxin (asassessed by the Limulus amebocyte assay), and were non-toxic inrecombinase-activating gene-2 (RAG-2^(−/−))-deficient mice, which arehighly susceptible to endotoxin shock (42). To ensure that the responseobserved in wild type mice was not due to TLR activation, MCP-1 and FKNresponse to IV sPDNF was assessed in TLR-deficient MyD88^(−/−) mice(23). Accordingly, the results showed that IV sPDNF gave rise to a sharpincrease in the expression of both MCP-1 and FKN in the heart and liverof MyD88^(−/−) mice (FIGS. 11E-11H). Without being bound to a particulartheory, this indicates that sPDNF acts on the Trk receptorsindependently of TLR signaling.

Example 7. Multiple Doses of IV sPDNF Increased Expression of MCP-1 andFKN Receptors CCR2 and CX3CR1 in the Mouse Heart and Liver

One of the functions of MCP-1 and FKN is to attract cells expressingtheir receptors (CCR2 and CX3CR1, respectively) to tissue sites.Following a single IV sPDNF dose (150 μg), CCR2 and CX3CR1 transcriptsdid not increase in cardiac and hepatic tissues (FIGS. 12A and 12B),consistent with the finding that MCP-1 and FKN increase was transientand pulse-like (FIG. 11A). However, sustained tissue exposure to sPDNFobtained by multiple and closely spaced systemic sPDNF injections intonaïve mice (IV sPDNF at times 0, 3 and 24 hr, measuring chemokines 24 hrafter the last injection), resulted in a significant increase in CCR2and CX3CR1 in the liver and heart (ventricle and atrium) (FIGS. 12C and12D). Thus, sPDNF-induced MCP-1 and FKN promoted migration of cellsbearing CCR2 and CX3CR1 receptors.

The results described herein were obtained using the following materialsand methods.

Purification of Recombinant sPDNF

PDNF was cloned from the T. cruzi Silvio X-10/4 strain (GenBankaccession number AJ002174). PDNF and a N-terminal short-form of PDNF(sPDNF) that contains Trk-binding sites, were expressed in BL21 (DE3)bacteria, and purified by Ni-affinity chromatography, as describedpreviously (4, 12, 18, 60). Buffers were prepared in sterileendotoxin-free water, and sPDNF preparations screened with the Limulusamebocyte assay have undetectable levels of endotoxin. sPDNF migrates asa 68 kDa protein on a SDS-PAGE gel. PDNF protein was buffer exchangedinto PBS (0.01 M phosphate buffered saline, pH 7.2), filter-sterilized(0.22 μm), and kept at 4° C. PDNF was quantified by scanningdensitometry (Bio-Rad, GS-800) of SDS-PAGE gels stained with Coomassiebrilliant blue.

Parasites

Experiments were performed with T. cruzi Tulahuen strain, which werepropagated in Vero cells. Free-swimming infective trypomastigotes wereharvested from supernatants (3-5 days after infection) by initial lowspeed centrifugation (500×g, 5 min) to remove host cells and debris,followed by high-speed centrifugation (1,200×g, 10 min) to pelletparasites, and resuspended in DMEM/0.1% FCS. For in vivo experiments,parasites were resuspended in PBS prior to injection into mice.

Mice

Female C57BL/6 mice, 6-8 weeks of age and C57BL/6 breeding pairs werefrom the Jackson Laboratory (Bar Harbor, Me.). MyD88-knockout mice werea generous gift from Dr. Thereza Imanishi-Kari, Tufts Medical School(21). All mouse experiments were approved by the Institutional AnimalCare and Use Committee (IACUC) and the Division of Laboratory AnimalMedicine (DLAM) of Tufts University School of Medicine.

Cell Lines and Primary Cell Cultures (i) Cell Lines.

H9c2 (ATCC® CRL-1446) (rat cardiomyocyte) and HEK 293 cells (usedgenerate lentiviral particles) were maintained in DMEM/10% FCS, and Verocells (used to grow T. cruzi) in DMEM/1% FCS. Cells were incubated at37° C. in a humidified atmosphere containing 5% CO₂.

(ii) Primary Cardiomyocytes and Cardiac Fibroblasts.

Cells were isolated using a modified version of a previously describedprocedure (4, 5, 51). In short, neonatal C56BL/6 mice (1-3 days old)were sacrificed by decapitation, their hearts excised and washed twicein PBS, then kept in 20 mM HEPES, 130 mM NaCl, 1 mM NaH₂PO₄, 4 mMglucose, 3 mM KCl, pH 7.6 for 10 minutes on ice. Hearts were minced anddigested in 0.25% Trypsin-EDTA (Gibco) 3-4 times at 37° C. with periodicmixing. Dissociated cells were pooled and digestion stopped withDMEM/10% FCS, filtered through a 100̂m cell strainer, centrifuged at500×g, and plated for 3 h on 1% gelatin-coated plates in DMEM(Gibco)/F12 Ham's (Sigma) 50:50, 20% FCS (PAA), 5% horse serum (PAA), 2mM L-glutamine (Gibco), 0.1 mM nonessential amino acids (Gibco), 3 mMsodium pyruvate (Gibco), and 1 Ag/ml bovine insulin (Sigma) with 1×penicillin-streptomycin (Gibco). Non-adherent cells (cardiomyocytes)were removed and plated onto new gelatin-coated plates while adherentcells (cardiac fibroblasts) remained in the initial plate in DMEM/10%FCS until needed. Cardiomyocytes contained <5% cardiac fibroblasts andvice versa for cardiac fibroblasts, as determined by immunofluorescenceusing antibodies against the cardiomyocyte marker myosin heavy chain(MHC) and cardiac fibroblast marker vimentin, as described earlier (4).

Cell Stimulation

(i) T. cruzi.

Primary cardiomyocytes and cardiac fibroblasts were seeded at 6-8×10⁴cells per well in DMEM/10% FCS and allowed to adhere overnight. Mediawas changed to DMEM/5% FCS and cells were infected with T. cruzi at anMOI (multiplicity of infection) of 10 for 10-24 h, at which timeparasites were washed off. At 3, 24, and 72 hr post-infection (PI), cellmonolayers were collected in Trizol reagent (Invitrogen) to isolate RNAto generate cDNA for qPCR.

(ii) sPDNF.

Primary cardiomyocytes and cardiac fibroblasts, and H9c2 cardiomyocyteswere seeded at 6-8×10⁴ cells per well in DMEM/10% FCS and allowed toadhere overnight, serum starved (DMEM/0.1% FCS overnight), and treatedwith sPDNF (0.5 ng/ml-8 μg/ml, 0-16 h). Cell supernatants were collectedand stored at −80° C. and cell monolayers rinsed with PBS and collectedin Trizol for qPCR analysis.

Blocking Trk Receptors (i) Pharmacological Inhibitor.

H9c2 cells were pre-treated with 0.1% DMSO vehicle control or 200 nMK252a for 1 h prior to addition of sPDNF (1 μg/ml). Supernatants andcell monolayers were collected for quantification of secreted chemokinesand cells were harvested for mRNA quantification by qPCR.

(ii) Neutralizing Antibodies.

H9c2 cardiomyocytes were pre-treated with 1 μg/ml neutralizingantibodies against TrkA (a-TrkA, Santa Cruz SC-118), TrkB (a-TrkB, SantaCruz SC-8316), and TrkC (a-TrkC, Santa Cruz SC-14025) for 30 min, andthen stimulated with 1 μg/ml sPDNF; chemokine transcripts werequantified by qPCR.

(iii) Lentiviral Transfection and Trk Knockdown by shRNA.

Lentiviral vectors encoding shRNA constructs targeting TrkA (clones1-4), TrkC (clones 1-5) or GFP mRNA (Open Biosystems) were generatedfrom transfected HEK 293 cells following manufacturer's instructions,and aliquots were frozen at −80° C. until use. Lentiviral infection ofH9c2 cells was performed after pretreatment with 8 μg/ml polybrene(Sigma-Aldrich) in DMEM/10% FCS, with a dose of 200 μl vector-containingsupernatant/2 ml medium in 6-well plates. Lentiviral particles wereremoved 24 hr after infection and, 7-8 d later, cells were serum starvedin serum-free DMEM for 2 hr and stimulated without or with sPDNF (4μg/ml, 3 hr).

Quantitative Real-Time PCR (qPCR)

RNA was isolated from Trizol lysates of cell monolayers or liquidnitrogen snap frozen tissue samples dissociated by a Tissue-Tearormechanical homogenizer (Biospec Products, Inc.). cDNA was synthesizedusing the Quantitect Reverse Transcription kit (Qiagen) according tomanufacturer's instructions. Chemokine and chemokine receptortranscripts were amplified using specific primers and normalized to HPRTusing SYBR Green (Qiagen), and, if needed, expressed relative tocontrol-unstimulated cells. The following primers were used:

MCP-1 (F: 5′-TCTCTTCCTCCACCACTATGCA-3′ (SEQ ID NO: 7);R: 5′-GGCTGAGACAGCACGTGGAT-3′ (SEQ ID NO: 8)), FKN(F: 5′-GCCCGCCGAATTCCTGCACT-3′ (SEQ ID NO: 9);R: 5′-CAATGGCACGCTTGCCGCAG-3′ (SEQ ID NO: 10)), CCR2(F: 5′-AATGAGAAGAAGAGGCACAGGGCT-3′ (SEQ ID NO: 11);R: 5′-ATGGCCTGGTCTAAGTGCTTGTCA-3′ (SEQ ID NO: 12)), CX3CR1(F: 5′-CGACATTGTGGCCTTTGGAACCAT-3′ (SEQ ID NO: 13);R: 5′-AGATGTCAGTGATGCTCTTGGGCT-3′ (SEQ ID NO: 14)), and HPRT(F: 5′-CAGCGTCGTGATTAGCGATGATG-3′ (SEQ ID NO: 15);R: 5′-CGAGCAAGTCTTTCAGTCCTGTC-3′ (SEQ ID NO: 16)).Enzyme-Linked Immunosorbent Assay (ELISA) MCP-1: 96-well Maxisorp plates(Nunc) were coated overnight with relevant supernatants or MCP-1standards (R&D Systems, 479-JE/CF) in coating buffer (50 mMNaHCO₃/Na₂CO₃, pH 9.6, 0.02% NaN₃), blocked in 5% BSA/PBST, reacted witha MCP-1 detection antibody (Santa Cruz, SC-28879; 1:200; 2 hr), and thenalkaline-phosphatase (AP) conjugated secondary antibody (Sigma A3687;1:1000; 1 hr). Between each step, plates were washed 2-4 times with PBST. Wells were then incubated with colorigenic AP substrate (SigmaN9389, 1 mg/ml in 100 mM glycine, 1 mM MgCl₂, 1 mM ZnCl₂, pH 10.4) andabsorbance read at 405 nm on an Emax precision microplate reader(Molecular Devices). MCP-1 concentrations were calculated relative to a4-parametric standard curve using the SOFTmax Pro program. MIP-2a (R&DSystems, DY452) and IL-6 (R&D Systems, DY406) ELISAs were done accordingto manufacturer's protocols.Mouse Model of Acute Chagas Disease and Intravenous (IV) sPDNFAdministration into Naive Mice

(i) Mouse Model.

Female C57BL/6 mice (6-8 weeks old) were infected subcutaneously in theleft hind footpad with 5×10³ trypomastigotes in 30 μl under isofluorineanesthesia, and sacrificed at various days post-infection (DPI) by CO₂asphyxiation and cervical dislocation. Organs were perfused byintracardiac injection of 5 ml ice-cold PBS, and the heart (sometimes,atria and ventricle were collected separately) and other organs werecollected and flash frozen in liquid nitrogen and stored at −80° C. orfixed in 4% paraformaldehyde for frozen sections. Tissue parasitism wasquantified using a qPCR method previously described (17, 18, 60).

(ii) IV Administration.

sPDNF was diluted in sterile endotoxin-free PBS (200 μl) and injectedvia the tail vein into 6-8 week old female C57BL/6 mice. Mice receivingsingle IV injections of 150 μg sPDNF per mouse were sacrificed at 3, 6,9, and 12 hr post-injection, or various doses of sPDNF (100 ng-150 μg)and sacrificed 3 hr post-injection. Mice receiving multiple injectionsof sPDNF or PBS vehicle control (0, 3, and 24 hr, 100 μg per injection)were sacrificed 24 hr after the last injection (i.e., 48 hr after thefirst injection). Mice were perfused with PBS and organs harvested asdescribed above.

Statistical Analyses

Statistical analysis was performed using GraphPad Prism software(version 5.0) using Student's/-test (for comparing two samples) orone-way ANOVA with Tukey's post-test (for comparing three or moresamples).

REFERENCES

-   1. Aliberti, J. C., M. A. Cardoso, G. A. Martins, R. T.    Gazzinelli, L. Q. Vieira, and J. S. Silva. 1996. Interleukin-12    mediates resistance to Trypanosoma cruzi in mice and is produced by    murine macrophages in response to live trypomastigotes. Infect Immun    64:1961-1967.-   2. Almeida, I. C., M. M. Camargo, D. O. Procopio, L. S. Silva, A.    Mehlert, L. R. Travassos, R. T. Gazzinelli, and M. A.    Ferguson. 2000. Highly purified glycosylphosphatidylinositols from    Trypanosoma cruzi are potent proinflammatory agents. EMBO J    19:1476-1485.-   3. Antunez, M. I., and R. L. Cardoni. 2000. IL-12 and IFN-gamma    production, and NK cell activity, in acute and chronic experimental    Trypanosoma cruzi infections. Immunol Lett 71:103-109.-   4. Aridgides, D., R. Salvador, and M. PereiraPerrin. 2013.    Trypanosoma cruzi Coaxes Cardiac Fibroblasts into Preventing    Cardiomyocyte Death by Activating Nerve Growth Factor Receptor TrkA.    PLoS ONE 8:e57450.-   5. Aridgides, D., R. Salvador, and M. Pereiraperrin. 2013.    Trypanosoma cruzi highjacks TrkC to enter cardiomyocytes and cardiac    fibroblasts while exploiting TrkA for cardioprotection against    oxidative stress. Cell Microbiol 15:1357-1366.-   6. Bastos, C. J., R. Aras, G. Mota, F. Reis, J. P. Dias, R. S. de    Jesus, M. S. Freire, E. G. de Araujo, J. Prazeres, and M. F.    Grassi. 2010. Clinical outcomes of thirteen patients with acute    chagas disease acquired through oral transmission from two urban    outbreaks in northeastern Brazil. PLoS Negl Trop Dis 4:e711.-   7. Bastos, K. R., R. Barboza, L. Sardinha, M. Russo, J. M. Alvarez,    and M. R. Lima. 2007. Role of endogenous IFN-gamma in macrophage    programming induced by IL-12 and IL-18. Journal of interferon &    cytokine research: the official journal of the International Society    for Interferon and Cytokine Research 27:399-410.-   8. Camargo, M. M., I. C. Almeida, M. E. Pereira, M. A.    Ferguson, L. R. Travassos, and R. T. Gazzinelli. 1997.    Glycosylphosphatidylinositol-anchored mucin-like glycoproteins    isolated from Trypanosoma cruzi trypomastigotes initiate the    synthesis of proinflammatory cytokines by macrophages. J Immunol    158:5890-5901.-   9. Chessler, A. D., L. R. Ferreira, T. H. Chang, K. A. Fitzgerald,    and B. A. Burleigh. 2008. A novel IFN regulatory factor 3-dependent    pathway activated by trypanosomes triggers IFN-beta in macrophages    and fibroblasts. J Immunol 181:7917-7924.-   10. Chuenkova, M. V., and M. A. Pereira. 2003. PDNF, a human    parasite-derived mimic of neurotrophic factors, prevents caspase    activation, free radical formation, and death of dopaminergic cells    exposed to the Parkinsonism-inducing neurotoxin MPP+. Brain Res Mol    Brain Res 119:50-61.-   11. Chuenkova, M. V., and M. A. Pereira. 2000. A trypanosomal    protein synergizes with the cytokines ciliary neurotrophic factor    and leukemia inhibitory factor to prevent apoptosis of neuronal    cells. Mol Biol Cell 11:1487-1498.-   12. Chuenkova, M. V., and M. PereiraPerrin. 2004. Chagas' disease    parasite promotes neuron survival and differentiation through TrkA    nerve growth factor receptor. J Neurochem 91:385-394.-   13. Chuenkova, M. V., and M. PereiraPerrin. 2005. A synthetic    peptide modeled on PDNF, Chagas' disease parasite neurotrophic    factor, promotes survival and differentiation of neuronal cells    through TrkA receptor. Biochemistry 44:15685-15694.-   14. Chuenkova, M. V., and M. PereiraPerrin. 2009. Trypanosoma cruzi    targets Akt in host cells as an intracellular antiapoptotic    strategy. Sci Signal 2:ra74.-   15. Chuenkova, M. V., and M. Pereiraperrin. 2010. Trypanosoma    cruzi-Derived Neurotrophic Factor: Role in Neural Repair and    Neuroprotection. Journal of neuroparasitology 1:55-60.-   16. Coelho, P. S., A. Klein, A. Talvani, S. F. Coutinho, O.    Takeuchi, S. Akira, J. S. Silva, H. Canizzaro, R. T. Gazzinelli,    and M. M. Teixeira. 2002. Glycosylphosphatidylinositol-anchored    mucin-like glycoproteins isolated from Trypanosoma cruzi    trypomastigotes induce in vivo leukocyte recruitment dependent on    MCP-1 production by IFN-gamma-primed-macrophages. J Leukoc Biol    71:837-844.-   17. Cummings, K. L., and R. L. Tarleton. 2003. Rapid quantitation of    Trypanosoma cruzi in host tissue by real-time PCR. Mol Biochem    Parasitol 129:53-59.-   18. de Melo-Jorge, M., and M. PereiraPerrin. 2007. The Chagas'    disease parasite Trypanosoma cruzi exploits nerve growth factor    receptor TrkA to infect mammalian hosts. Cell host & microbe    1:251-261.-   19. Frangogiannis, N. G. 2012. Matricellular proteins in cardiac    adaptation and disease. Physiological reviews 92:635-688.-   20. Goncalves, V. M., K. C. Matteucci, C. L. Buzzo, B. H. Miollo, D.    Ferrante, A. C. Torrecilhas, M. M. Rodrigues, J. M. Alvarez,    and K. R. Bortoluci. 2013. NLRP3 controls Trypanosoma cruzi    infection through a caspase-1-dependent IL-1R-independent NO    production. PLoS Negl Trop Dis 7:e2469.-   21. Han, J. H., S. Akira, K. Calame, B. Beutler, E. Selsing, and T.    Imanishi-Kari. 2007. Class switch recombination and somatic    hypermutation in early mouse B cells are mediated by B cell and    Toll-like receptors. Immunity 27:64-75.-   22. Huang, E. J., and L. F. Reichardt. 2003. Trk receptors: roles in    neuronal signal transduction. Annu Rev Biochem 72:609-642.-   23. Kawai, T., and S. Akira. 2007. TLR signaling. Seminars in    immunology 19:24-32.-   24. Koga, R., S. Hamano, H. Kuwata, K. Atarashi, M. Ogawa, H.    Hisaeda, M. Yamamoto, S. Akira, K. Himeno, M. Matsumoto, and K.    Takeda. 2006. TLR-dependent induction of IFN-beta mediates host    defense against Trypanosoma cruzi. J Immunol 177:7059-7066.-   25. Machado, F. S., W. O. Dutra, L. Esper, K. J. Gollob, M. M.    Teixeira, S. M. Factor, L. M. Weiss, F. Nagajyothi, H. B. Tanowitz,    and N. J. Garg. 2012. Current understanding of immunity to    Trypanosoma cruzi infection and pathogenesis of Chagas disease.    Seminars in immunopathology 34:753-770.-   26. Machado, F. S., G. A. Martins, J. C. Aliberti, F. L.    Mestriner, F. Q. Cunha, and J. S. Silva. 2000. Trypanosoma    cruzi-infected cardiomyocytes produce chemokines and cytokines that    trigger potent nitric oxide-dependent trypanocidal activity.    Circulation 102:3003-3008.-   27. Marin-Neto, J. A., E. Cunha-Neto, B. C. Maciel, and M. V.    Simoes. 2007. Pathogenesis of chronic Chagas heart disease.    Circulation 115:1109-1123.-   28. Martins, R. F., P. M. Martinelli, P. M. Guedes, B. da Cruz    Padua, F. M. Dos Santos, M. E. Silva, M. T. Bahia, and A.    Talvani. 2013. Protein deficiency alters CX3CL1 and endothelin-1 in    experimental Trypanosoma cruzi-induced cardiomyopathy. Tropical    medicine & international health: TM & IH 18:466-476.-   29. Michailowsky, V., N. M. Silva, C. D. Rocha, L. Q. Vieira, J.    Lannes-Vieira, and R. T. Gazzinelli. 2001. Pivotal role of    interleukin-12 and interferon-gamma axis in controlling tissue    parasitism and inflammation in the heart and central nervous system    during Trypanosoma cruzi infection. Am J Pathol 159:1723-1733.-   30. Morganti, J. M., K. R. Nash, B. A. Grimmig, S. Ranjit, B.    Small, P. C. Bickford, and C. Gemma. 2012. The soluble isoform of    CX3CL1 is necessary for neuroprotection in a mouse model of    Parkinson's disease. J Neurosci 32:14592-14601.-   31. Morimoto, H., M. Hirose, M. Takahashi, M. Kawaguchi, H.    Ise, P. E. Kolattukudy, M. Yamada, and U. Ikeda. 2008. MCP-1 induces    cardioprotection against ischaemia/reperfusion injury: role of    reactive oxygen species. Cardiovascular research 78:554-562.-   32. Morimoto, H., M. Takahashi, A. Izawa, H. Ise, M. Hongo, P. E.    Kolattukudy, and U. Ikeda. 2006. Cardiac overexpression of monocyte    chemoattractant protein-1 in transgenic mice prevents cardiac    dysfunction and remodeling after myocardial infarction. Circ Res    99:891-899.-   33. Nahrendorf, M., F. K. Swirski, E. Aikawa, L. Stangenberg, T.    Wurdinger, J. L. Figueiredo, P. Libby, R. Weissleder, and M. J.    Pittet. 2007. The healing myocardium sequentially mobilizes two    monocyte subsets with divergent and complementary functions. J Exp    Med 204:3037-3047.-   34. Paiva, C. N., R. T. Figueiredo, K. Kroll-Palhares, A. A.    Silva, J. C. Silverio, D. Gibaldi, S. Pyrrho Ados, C. F.    Benjamim, J. Lannes-Vieira, and M. T. Bozza. 2009. CCL2/MCP-1    controls parasite burden, cell infiltration, and mononuclear    activation during acute Trypanosoma cruzi infection. J Leukoc Biol    86:1239-1246.-   35. Parada, H., H. A. Carrasco, N. Anez, C. Fuenmayor, and I.    Inglessis. 1997. Cardiac involvement is a constant finding in acute    Chagas' disease: a clinical, parasitological and histopathological    study. Int J Cardiol 60:49-54.-   36. Parodi, A. J., G. D. Pollevick, M. Mautner, A. Buschiazzo, D. O.    Sanchez, and A. C. Frasch. 1992. Identification of the gene(s)    coding for the trans-sialidase of Trypanosoma cruzi. EMBO J    11:1705-1710.-   37. Pereira, M. E. 1983. A developmentally regulated neuraminidase    activity in Trypanosoma cruzi. Science 219:1444-1446.-   38. Pereira, M. E., M. A. Loures, F. Villalta, and A. F.    Andrade. 1980. Lectin receptors as markers for Trypanosoma cruzi.    Developmental stages and a study of the interaction of wheat germ    agglutinin with sialic acid residues on epimastigote cells. J Exp    Med 152:1375-1392.-   39. Pinto, A. Y., A. G. Ferreira, Jr., C. Valente Vda, G. S. Harada,    and S. A. Valente. 2009. Urban outbreak of acute Chagas disease in    Amazon region of Brazil: four-year follow-up after treatment with    benznidazole. Revista panamericana de salud publica=Pan American    journal of public health 25:77-83.-   40. Prioli, R. P., J. S. Mejia, T. Aji, M. Aikawa, and M. E.    Pereira. 1991. Trypanosoma cruzi: localization of neuraminidase on    the surface of trypomastigotes. Trop Med Parasitol 42:146-150.-   41. Rassi, A., Jr., A. Rassi, and J. A. Marin-Neto. 2010. Chagas    disease. Lancet 375:1388-1402.-   42. Reid, R. R., A. P. Prodeus, W. Khan, T. Hsu, F. S. Rosen,    and M. C. Carroll. 1997. Endotoxin shock in antibody-deficient mice:    unraveling the role of natural antibody and complement in the    clearance of lipopolysaccharide. J Immunol 159:970-975.-   43. Rodrigues, A. A., J. S. Saosa, G. K. da Silva, F. A.    Martins, A. A. da Silva, C. P. Souza Neto, C. V. Horta, D. S.    Zamboni, J. S. da Silva, E. A. Ferro, and C. V. da Silva. 2012.    IFN-gamma plays a unique role in protection against low virulent    Trypanosoma cruzi strain. PLoS Negl Trop Dis 6:e1598.-   44. Schauer, R., G. Reuter, H. Muhlpfordt, A. F. Andrade, and M. E.    Pereira. 1983. The occurrence of N-acetyl- and N-glycoloylneuraminic    acid in Trypanosoma cruzi. Hoppe Seylers Z Physiol Chem    364:1053-1057.-   45. Schenkman, S., L. Pontes de Carvalho, and V. Nussenzweig. 1992.    Trypanosoma cruzi trans-sialidase and neuraminidase activities can    be mediated by the same enzymes. J Exp Med 175:567-575.-   46. Scudder, P., J. P. Doom, M. Chuenkova, I. D. Manger, and M. E.    Pereira. 1993. Enzymatic characterization of beta-D-galactoside    alpha 2,3-trans-sialidase from Trypanosoma cruzi. J Biol Chem    268:9886-9891.-   47. Sebastiani, S., P. Allavena, C. Albanesi, F. Nasorri, G.    Bianchi, C. Traidl, S. Sozzani, G. Girolomoni, and A. Cavani. 2001.    Chemokine receptor expression and function in CD4+T lymphocytes with    regulatory activity. J Immunol 166:996-1002.-   48. Serbina, N. V., T. Jia, T. M. Hohl, and E. G. Pamer. 2008.    Monocyte-mediated defense against microbial pathogens. Annual review    of immunology 26:421-452.-   49. Sheridan, G. K., and K. J. Murphy. 2013. Neuron-glia crosstalk    in health and disease: fractalkine and CX3CR1 take centre stage.    Open biology 3:130181.-   50. Shibayama, E., and H. Koizumi. 1996. Cellular localization of    the Trk neurotrophin receptor family in human non-neuronal tissues.    Am J Pathol 148:1807-1818.-   51. Sreejit, P., S. Kumar, and R. S. Verma. 2008. An improved    protocol for primary culture of cardiomyocyte from neonatal mice. In    vitro cellular & developmental biology. Animal 44:45-50.-   52. Tamura, Y., K. Matsumura, M. Sano, H. Tabata, K. Kimura, M.    leda, T. Arai, Y. Ohno, H. Kanazawa, S. Yuasa, R. Kaneda, S.    Makino, K. Nakajima, H. Okano, and K. Fukuda. 2011. Neural    crest-derived stem cells migrate and differentiate into    cardiomyocytes after myocardial infarction. Arterioscler Thromb Vasc    Biol 31:582-589.-   53. Tapley, P., F. Lamballe, and M. Barbacid. 1992. K252a is a    selective inhibitor of the tyrosine protein kinase activity of the    trk family of oncogenes and neurotrophin receptors. Oncogene    7:371-381.-   54. Teixeira, M. M., R. T. Gazzinelli, and J. S. Silva. 2002.    Chemokines, inflammation and Trypanosoma cruzi infection. Trends    Parasitol 18:262-265.-   55. Truyens, C., A. Angelo-Barrios, F. Torrico, J. Van Damme, H.    Heremans, and Y. Carlier. 1994. Interleukin-6 (IL-6) production in    mice infected with Trypanosoma cruzi: effect of its paradoxical    increase by anti-IL-6 monoclonal antibody treatment on infection and    acute-phase and humoral immune responses. Infect Immun 62:692-696.-   56. Unnikrishnan, M., and B. A. Burleigh. 2004. Inhibition of host    connective tissue growth factor expression: a novel Trypanosoma    cruzi-mediated response. FASEB J 18:1625-1635.-   57. Wahab, N. A., B. S. Weston, and R. M. Mason. 2005. Connective    tissue growth factor CCN2 interacts with and activates the tyrosine    kinase receptor TrkA. J Am Soc Nephrol 16:340-351.-   58. Wang, X., S. V. McLennan, T. J. Allen, and S. M. Twigg. 2010.    Regulation of pro-inflammatory and pro-fibrotic factors by CCN2/CTGF    in H9c2 cardiomyocytes. Journal of cell communication and signaling    4:15-23.-   59. Weinkauf, C., and M. PereiraPerrin. 2009. Trypanosoma cruzi    promotes neuronal and glial cell survival through the neurotrophic    receptor TrkC. Infect Immun 77:1368-1375.-   60. Weinkauf, C., R. Salvador, and M. PereiraPerrin. 2011.    Neurotrophin receptor TrkC is an entry receptor for Trypanosoma    cruzi in neural, glial, and epithelial cells. Infect Immun    79:4081-4087.-   61. Zangi, L., K. O. Lui, A. von Gise, Q. Ma, W. Ebina, L. M.    Ptaszek, D. Spater, H. Xu, M. Tabebordbar, R. Gorbatov, B. Sena, M.    Nahrendorf, D. M. Briscoe, R. A. Li, A. J. Wagers, D. J.    Rossi, W. T. Pu, and K. R. Chien. 2013. Modified mRNA directs the    fate of heart progenitor cells and induces vascular regeneration    after myocardial infarction. Nat Biotechnol 31:898-907.

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

This application may be related to one or more of: U.S. patentapplication Ser. No. 13/345,210, filed on Jan. 6, 2012, abandoned, whichis a continuation of application Ser. No. 11/365,743, filed Feb. 28,2006, U.S. Pat. No. 8,114,412, which is a divisional of application Ser.No. 09/745,008, filed Dec. 20, 2000, U.S. Pat. No. 7,060,676, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/172,881,filed Dec. 20, 1999; U.S. patent application Ser. No. 13/505,316, filedon May 1, 2012, which is the U.S. national phase application, pursuantto 35 U.S.C. §371, of International Patent Application No.:PCT/US2010/055700, filed Nov. 5, 2010, which claims the benefit of U.S.Provisional Application Ser. No. 61/258,961, filed Nov. 6, 2009; U.S.patent application Ser. No. 11/982,371, filed on Nov. 1, 2007,abandoned, which claims the benefit of U.S. Provisional Application Ser.No. 60/856,170, filed Nov. 2, 2006. The disclosures of theaforementioned applications are hereby incorporated herein in theirentireties by reference.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1.-9. (canceled)
 10. A method of increasing stem cell number, stem cellmobilization, and/or stem cell proliferation of stem cells within anon-neuronal tissue or organ, the method comprising: contacting thenon-neuronal tissue or organ with a polypeptide of SEQ ID NO: 2, whichbinds to the TrkA receptor and/or the TrkC receptor on non-neuronalcells or tissue, or a TrkA receptor binding portion of the polypeptideof SEQ ID NO: 2 selected from residues 1-588, residues 33-666, orresidues 425-455 of SEQ ID NO: 2 in an amount effective to increase stemcell number, stem cell mobilization, and/or stem cell proliferation ofstem cells within the non-neuronal tissue or organ. 11.-12. (canceled)13. The method of claim 10, wherein the stem cell expresses Sca-1 and/orc-Kit.
 14. The method of claim 10, wherein the stem cell is a cardiac,hepatic, pancreatic, and/or gastrointestinal stem cell.
 15. The methodof claim 10, wherein the non-neuronal tissue or organ is a cardiac,hepatic, pancreatic, and/or gastrointestinal tissue or organ.
 16. Themethod of claim 10, wherein the stem cell is in vivo or ex vivo.
 17. Themethod of claim 16, wherein the stem cell is in a non-neuronal tissue ororgan in a subject.
 18. The method of claim 17, wherein the subject hasor is at risk of having an inflammatory disease.
 19. The method of claim18, wherein the inflammatory disease is a cardiac inflammatory disease,hepatic inflammatory disease, pancreatic inflammatory disease, orinflammatory disease of the gastrointestinal (GI) tract.
 20. The methodof claim 18, wherein the inflammatory disease is myocarditis,cardiomyopathy, endocarditis, pericarditis, hepatitis, cirrhosis,inflammatory bowel diseases (IBD), irritable bowel syndrome, ileitis,chronic inflammatory intestinal disease, or celiac disease, Crohn'sdisease, ulcerative colitis, type 1 diabetes, and/or type 2 diabetes.21.-25. (canceled)
 26. The method of claim 18, wherein ananti-inflammatory agent is administered to the subject. 27.-40.(canceled)